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Wound induced plant phenolic compounds and virulence gene expression in Agrobacterium species Spencer, Paul Anthony 1991

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W O U N D I N D U C E D P L A N T P H E N O L I C C O M P O U N D S A N D V I R U L E N C E G E N E E X P R E S S I O N I N A G R O B A C T E R I U M SPECIES by P A U L A N T H O N Y S P E N C E R B.Sc , The University of Victoria, 1985 M . S c , The University of British Columbia , 1988 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F " ^ . T H E R E Q U I R E M E N T S FOR T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y i n T H E F A C U L T Y O F G R A D U A T E STUDIES (Department of Botany) We accept this thesis as conforming to the required standard: T H E U N I V E R S I T Y O F BRITISH C O L U M B I A A p r i l 1991 © Paul Anthony Spencer, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) Abstract C r o w n gall disease of plants is caused by introduction of foreign D N A into susceptible plant cells by strains of Agrobacterium tumefaciens. The expression of bacterial virulence genes is triggered by chemicals present i n plant w o u n d exudates. The exudates contain a number of phenol ic compounds which act as chemical signals inducing expression of a number of genes directing the D N A transfer process. These are the virulence or vir genes, and virv.lac reporter gene fusions have been widely used to assay vir gene induct ion i n Agrobacterium tumefaciens strains. U s i n g such strains to monitor vir gene expression, Stachel et al. (1985) isolated from Nicotiana tabacum two active acetophenones: 3,5-dimethoxy-4-hydroxyacetophenone, ("acetosyringone" or A S ) , and a - h y d r o x y - 3 , 5 - d i m e t h o x y - 4 - h y d r o x y -acetophenone, ("hydroxyacetosyringone" or H O - A S ) . H o w e v e r , in vitro assay results suggested that other more common compounds also exhibited activity (Spencer and Towers, 1988). This analysis of structure-activity relationships of induced vir expression i n A. tumefaciens was presented i n a previous thesis (Paul Spencer, M.Sc. thesis). The results revealed that a variety of commonly occurring plant phenolic compounds were capable of activating vir genes. In addition to the acetophenones, a variety of benzoic and cinnamic acid derivatives, and even a few chalcones of appropriate r ing substitution were active. This thesis reports the isolation and identification of a number of these compounds in plant w o u n d exudates. Some Agrobacterium tumefaciens strains are restricted i n host range to certain grapevine cultivars. Subsequent to the development of a convenient and sensitive plate-bioassay method, a strongly active component i n grapevine w o u n d exudates was puri f ied. A newly described t ;zr- inducing phenolic compound was isolated from a number of Vitis cultivars using gel ii f i l tration, thin layer and high pressure l i q u i d chromatographies. This was identified as syringic acid methyl ester (3,5-dimethoxy-4-hydroxybenzoic acid, methy l ester), us ing mass spectrometry. However , the presence of this c o m p o u n d i n grapevine w o u n d exudates does not p r o v i d e a s imple explanation for host range limitation of grapevine strains since it induces vir gene expression i n both l imi ted and w i d e host range strains of A. tumefaciens. Interestingly, neither A S nor H O - A S were present i n grapevine-derived extracts. A convenient po lyamide c o l u m n chromatographic method was subsequently developed to permit rapid purification of plant-derived vir gene i n d u c i n g mixtures, which were detected using the newly developed plate bioassay. Derivatized polyamide fractions were then analysed by combined gas chromatography-mass spectrometry (GC-MS) . G C - M S proved to be an ideal means for the identification of the phenolic components i n partial ly puri f ied extracts. Examination of w o u n d exudates from a range of host and non-host species revealed that the product ion of the acetophenones is restricted to members of the Solanaceae. Some experiments focussed on the biosynthetic precursors of the acetophenones i n Nicotiana species. W o u n d exudates of the majority of species belonging to other plant families contained benzaldehydes and/or benzoic and cinnamic acid derivatives. The induct ion of virE gene expression was examined i n the related Agrobacterium species, A. rhizogenes. To do this, the virE::lacZ gene fusion p l a s m i d pSM358cd was introduced into A. rhizogenes A 4 by triparental mat ing and the strain "A4/pSM358cd" was used to analyze vir activation. Acetophenones, chalcones, benzaldehydes, and benzoic and cinnamic acid derivatives were found to activate vir genes i n A. rhizogenes. i i i Table of contents Abstract ; i i Table of contents i v List of abbreviations v i List of Tables ix List of Figures x Acknowledgements x i i Chapter 1. General Introduction 1.1 The biology of Agrobacterium tumefaciens 1 1.2 Signal molecules inducing gene expression i n the Rhizobiaceae 17 1.2.1 Structure-activity analysis of A. tumefaciens vir gene expression 17 1.2.2 Flavonoid nod gene inducers of Rhizobium species 27 1.3 Phenolic biochemistry of wounded plant tissues i n relation to vir gene expression 31 1.3.1 Phytoalexins .' 34 1.3.2 Ce l l wall-bound phenolics 36 1.3.3 Lignification 39 1.3.4 Suberization 42 1.3.5 De novo biosynthesis of phenolic compounds 44 1.3.6 Vacuolar phenolics 48 1.3.7 vir -repressors 51 1.4 Concluding remarks 53 Chapter 2. Identification of an A. tumefaciens signal compound from grapevine cultivars 2.1 Foreword 54 2.2 Introduction 54 2.3 Methods 57 2.3.1 Plant material. . 57 2.3.2 Extraction and Isolation 57 2.3.3 p/r-Induction assays 58 2.3.4 Bacterial strains, media and plasmids 58 2.4 Results and Discussion 59 Chapter 3. Natural ly occurring wide host range A. tumefaciens signal molecules 3.1 Foreword 65 3.2 Introduction 65 iv 3.3 Methods , 68 3.3.1 znr-Induction assays 68 3.3.2 Plant materials 69 3.3.3 H P L C of maize hydroxamates 69 3.3.4 Bioassay sample preparation 71 3.3.5 Polyamide V L C chromatography 71 3.3.6 Hydrolysis of glycosides 72 3.3.7 Cycloheximide treatment 73 3.3.8 Cellulase-induced release of inducers from tobacco cell walls •• 73 3.4 Results and Discussion 3.4.1 Signal compound arrays 74 3.4.2 Solanaceous t?ir-inducers 77 3.4.3 Biosynthetic precursors of acetophenones in Nicotiana silvestris 79 3.4.3.1 Glycosides as biosynthetic precursors 79 3.4.3.2 Effects of cycloheximide 80 3.4.3.3 Cellulase treated cell wal l material 81 3.4.4 Inducers and inhibitors from monocots 82 3.4.5 Conifer extracts 83 3.4.6 p H effects 84 Chapter 4. ui'r-expression i n A. rhizogenes A 4 4.1 Introduction 87 4.2 Methods 89 4.2.1Triparental mating 89 4.2.2 pir-induction assays 90 4.3 Results and Discussion 90 Chapter 5. GC-mass spectrometry of ptr-inducing mixtures 5.1 Introduction 103 5.2 Methods 104 5.2.1 Derivatization 104 5.2.2 G C conditons 104 5.2.3 Mass spectrometer ? 105 5.3 Results and Discussion 105 Chapter 6. General Conclusions 6.1 Survey of naturally occurring signal compounds 127 6.2 Glucosides and the effect of cycloheximide 130 6.3 p H changes in conditioned media 131 6.4 vir expression i n Agrobacterium rhizogenes A 4 / p S M 3 5 8 c d 132 References 134 v List of abbreviations Bacterial strains A348 A. tumefaciens strain 348 (a C58 derivative w i t h W H R pTiA6) A 3 4 8 / p S M A. tumefaciens strain 348 possessing virv.lac insertion p lasmid A856 A. tumefaciens strain 856 (a C58 derivative wi th L H R pTiAgl62) A 8 5 6 / p S M A. tumefaciens strain 856 possessing virv.lac insertion p lasmid C58 W H R A. tumefaciens strain wi th nopaline-type T i p lasmid JC2926 E. coli strain (provided by Dr. E.W. Nester, U.W.) JC2926/pRK E. coli strain possessing helper plasmid JC2926/pSM E. coli strain possessing virv.lac insertion plasmid L H R l imited host range A. tumefaciens strain (eg. A348) W H R wide host range A. tumefaciens strain (eg. A856) Plasmids p R i A. rhizogenes root inducing plasmid p R i A 4 A. rhizogenes strain A 4 root inducing plasmid pRK2013 triparental mating helper plasmid p S M virv.lac insertion plasmid p T i A. tumefaciens tumor inducing plasmid p T i A 6 A. tumefaciens strain A 6 (WHR) tumor inducing plasmid p T i A g l 6 2 A. tumefaciens strain A g l 6 2 (LHR) tumor inducing plasmid p V C K cloning vehicle with portions of p T i A 6 vir region Genes and genetic elements abg cellobiase gene of A. tumefaciens att attachment gene of A. tumefaciens eel cellulose gene of A. tumefaciens chv chromosomal virulence gene of A. tumefaciens lacZ B-galactosidase gene of E. coli nod nodulation gene of Rhizobium species psc polysaccharide gene of A. tumefaciens tzs zeatin gene of A. tumefaciens vir virulence gene of A. tumefaciens virv.lac transcriptional and translational gene fusion between vir locus and lacZ T - D N A transferred D N A of T i plasmids T n 3 : : H o H o l transposable genetic element for creation of transcriptional and translation gene fusions wi th lacZ v i Chemicals and other abbreviations 1 H - N M R proton nuclear magnetic resonance spectrum or spectrometry 4CL 4-coumarate Co A ligase 1 3 C - N M R carbon nuclear magnetic resonance spectrum or spectrometry A B A abscisic acid A S 3,5-dimethoxy-4-hydroxyacetophenone (acetosyringone) A V 4-hydroxy-3-methoxyacetophenone (acetovanillone) B F 3 boron trifluoride B S T F A bis(trimethylsilyl)trifluoroacetamide B u O A c butyl acetate Q - C i benzoic acid derivative (ring plus one carbon) Q - C 2 acetophenone derivative (ring plus two carbons) Q - C 3 phenylpropanoid derivative (ring plus three carbons) C A 4 H cinnamate 4-hydoxylase carb carbenicil l in carb1- carbenicillin resistant CDCI3 deuterated chloroform CH2CI2 dichloromethane C 1 C H 2 C H 2 C 1 dichloroethane CHCI3 ch loroform D I B O A 2,4-dihydroxy-2H-l,4,-benzoxazin-3(4H)-one D I M 2 B O A 2,4-dihydroxy-6,7-dimethoxy-2H-l,4,-benzoxazin-3(4H)-one D I M B O A 2,4-dihy droxy-7-metyhoxy-2H-l ,4,-benzoxazin-3(4H)-one D M S O dimethylsulfoxide EIMS electron impact mass spectrum E t O A c ethyl acetate F A X X O-[5-0-(frfl«s-feruloyl)-a-L-arabinofuranosyl]-(l-3)-O-f3-D-xylopyranosyl-(l-4)-D-xylanopyranose G C - E I M S gas chromatography-electron impact mass spectrometry H O - A S a-hydroxy-3,5-dimethoxy-4-hydroxyacetophenone, (a-hydroxyacetosyringone) H O - A V a-hydroxy-3-methoxy-4-hydroxyacetophenone, (a-hydroxyacetovanillone) H O F m formic acid H P L C high pressure l i q u i d chromatography H R - E I M S high resolution electron impact mass spectrometry kan kanamycin k a n r kanamycin resistant L B Lur ia broth LH-20 sephadex gel permeation chromatographic support L i A l H l i t h i u m a luminum hydride M + molecular ion M 2 B O A 6,7-dimethoxy-2-(3H)-benzoxazolinone m/z mass over charge ratio v i i M B O A 6-methoxy-2-(3H)-benzoxazolinone M e O H methanol M S Murashige and Skoog plant cell culture medium nal nalidixic acid n a l r nalidixic acid resistant N a O M e sodium methoxide n - B u O H 1-butanol P A L phenylalanine ammonia lyase P D potato dextrose P D A potato dextrose agar Rf retention factor used i n T L C R t retention time used i n G C SE-54 fused silica capillary column used in G C spec spectinomycin spec1" spectinomycin resistant T L C thin layer chromatography T M S trimethylsilyl group (C3HioSi, m.w. 73) V L C vacuum l iquid chromatography X-gal 5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside v i i i List of Tables Table 2.1 Spectral and chromatographic data used i n the identification of methylsyringate i n grapevine w o u n d exudates 61 Table 3.1 Species examined for CHCl3-soluble znr-inducers 70 Table 3.2 The signalling phenolics detected by G C - M S of w o u n d exudates from selected species of flowering plants 76 Table 3.3 Final p H of conditioned media from selected plant species 86 Table 5.1 Phenolic compounds identified by G C - M S 121 Table 5.2 Mass spectral data for p/r-inducing phenolics 122 Table 5.3 Commonly observed fatty acids i n air-inducing mixtures 125 Table 5.4 pfr-inducing phenolics listed by m.w. , M + , and scan # 126 ix List of Figures Figure 1.1 Typical organization of the vir region of a Ti plasmid 7 Figure 1.2 Strategy for analysis of signal compound inducible p T i genes 10 Figure 1.3 Structures of seven phenolic compounds examined for vir -induction by Bolton et al. (1986) 11 Figure 1.4 The structures of acetosyringone (AS) and a-hydroxyacetosyringone (HO-AS) 13 Figure 1.5 The 16 chemicals used i n the study of structure-activity relationships, arranged into 4 classes 19 Figure 1.6 virE expression i n A. tumefaciens i n response to phenolic compounds 20 Figure 1.7 The structures nod-inducing flavonoids recently identified from host plants 28 Figure 1.8 Authentic flavonoids used i n the analysis of R. leguminosarum n o d - i n d u c t i o n 30 Figure 1.9 Formation of cell wall-bound diferulate 37 Figure 1.10 Structure of F A X X : 38 Figure 1.11 Generalized structure of suberin 43 Figure 1.12 Ethylene-induced isocoumarins and chromones 45 Figure 1.13 Biosynthetic pathway to A S and Me-syringate 47 Figure 1.14 Structures of acetophenone glucosides picein, pungenin, and androsin 50 Figure 1.15 D I M B O A and related structures 52 Figure 2.1 virB-induction i n strains A348 (WHR) and A856 (LHR) 63 Figure 4.1 Diagramatic illustration of triparental mating experiment to generate an Agrobacterium rhizogenes strain possessing a vir::lac insertion plasmid 88 Figure 4.2 vir gene activation i n A4/pSM358cd by acetophenones 92 Figure 4.3 vir gene activation i n A4/pSM358cd by phenolic acids 93 Figure 4.4 vir gene activation in A4/pSM358cd by benzaldehydes 94 Figure 4.5 vir gene activation i n A4/pSM358cd by coniferyl alcohol 95 Figure 4.6 vir gene activation in A4/pSM358cd by 2',4',4-trihydroxy-3-methoxy chalcone 96 Figure 4.7 vir gene activation i n A4/pSM358cd by methyl esters 97 Figure 5.1 Mass spectrum of 3,5-dimethoxy-4-hydroxyacetophenone (TMS derivative) 104 Figure 5.2 Mass spectrum of 3-methoxy-4-hydroxyacetophenone (TMS derivative) 105 x Figure 5.3 Mass spectrum of a-hydroxy-3,5-dimethoxy-4-hydroxyacetophenone (TMS derivative) 106 Figure 5.4 Mass spectrum of a-hydroxy-3-methoxy-4-hydroxyacetophenone (TMS derivative) 107 Figure 5.5 Mass spectrum of 3,5-dimethoxy-4-hydroxycinnamic acid (TMS derivative) 108 Figure 5.6 Mass spectrum of 3-methoxy-4-hydroxycinnamic acid (TMS derivative) 109 Figure 5.7 Mass spectrum of 3,5-dimethoxy-4-hydroxybenzoic acid (TMS derivative) 110 Figure 5.8 Mass spectrum of 3-methoxy-4-hydroxybenzoic acid (TMS derivative) I l l Figure 5.9 Mass spectrum of methyl-3,5-dimethoxy-4-hydroxybenzoic acid (TMS derivative) 112 Figure 5.10 Mass spectrum of methyl-3-methoxy-4-hydroxybenzoic acid (TMS derivative) 113 Figure 5.11 Mass spectrum of coniferyl alcohol (TMS derivative) 114 Figure 5.12 Keto-enol tautomerism of acetophenones ..116 Figure 5.13 Mass spectrum of A S enol tautomer (TMS derivative) 117 Figure 5.14 Mass spectrum of A V enol tautomer (TMS derivative) 118 Figure 6.1 Hypothesized pathway to the acetophenones i n Solanaceous plants 129 x i Acknowledgements I thank all those who contributed to this thesis. I am deeply indebted to D r . Eugene Nester (University of Washington) who provided the required Agrobacterium tumefaciens and other bacterial strains. It was m y privilege to have had as m y supervisor Dr . G . H . N . (Neil) Towers and to have worked alongside the people i n his lab: Zyta A b r a m o w s k i , Felipe Balza, N e s r i n Tanrisever, M i n o r u Terasawa, Hector Barrios, E d Neeland, John M c C a l l u m , D o n Champagne, and Shona El l is . Not forgotten are the numerous and useful discussions w i t h interested graduate students and others i n the Department of Botany. I thank Drs. Joan McPherson for her continuing interest i n this project and an excellent introduction to plant genetics and "agrobacteriology", and C a r l Douglas and Tony Warren for their m u c h valued input. Dr. A k i r a Tanaka (Plantec Research Institute, Japan) assisted i n the prel iminary work on the isolation of the grapevine inducer, for w h i c h I am grateful. I thank Dr. Bruce A . Bohm for providing rare chemicals and for discussions concerning natural products chemistry and methods of analysis. Thanks are due to m y parents for their continuing support . Financial support , i n the form of Teaching Assistantships, U n i v e r s i t y Graduate Fel lowships , and Research Assistantships (from N e i l Towers) is gratefully acknowledged. Thanks are due also to the Department of Chemistry Mass Spectrometry Facility, the Agriculture Canada Research Station at U . B . C . for p r o v i d i n g a variety of Nicotiana species, Dr. Gerald Straley for specimens from the U . B . C . Botanical Gardens, and Dr. George Eaton (Plant Science) for supplying plant material from the South Campus vineyard. x i i Chapter 1. General Introduction 1.1 The biology of A. tumefaciens In recent times there have been significant advances i n research concerning the interactions between plants and microorganisms. In part, this is because the techniques of molecular biology have al lowed us to probe the genetic bases of such relationships. Perhaps nowhere else has this type of analysis progressed so rapidly as wi th the study of crown gall tumorigenesis. It turns out, however, that a complete understanding of the interaction of Agrobacterium tumefaciens wi th plants requires also an understanding of the biochemical reactions triggered at the site of plant wounds. In comparison w i t h topics such as the biosynthesis of phytoalexins, this is a relatively uncharted domain. Initiation of plant-microbe relationships often requires exchange of chemical signals (reviewed by Dixon and Lamb, 1990). The microbe detects host-derived chemicals that are produced under certain conditions (eg. wounding) , or w h i c h the plant already possesses as part of its cell w a l l or membrane. The microbe may respond to these signals through spore germination (Fries et al, 1987), directed growth or chemotaxis (Ashby et al., 1987), and expression of genes necessary for subsequent stages of the interaction (Stachel et al, 1985b). For example, expression of genes directing morphogenesis f rom saprophytic to parasitic stages i n response to cc-tocopherol is k n o w n i n Ustilago violacea (Castle and Day, 1984). A t present, there are only a few examples of cell-cell signall ing between plants and microbes i n w h i c h the chemicals that induce such responses have been identified. However , the signal molecules mediating early stages of certain 1 bacterial-plant interactions have been identified (Stachel et al, 1985b; Peters et al, 1986; Redmond et al, 1986; F i rmin et al, 1986; Sadowsky et al, 1988; Spencer et al, 1990: Morr is and Morr is , 1990). These studies have focussed on compounds emanating from host plants and to which species of bacteria belonging to the Rhizobiaceae respond. This group of microbes includes the l egume-nodula t ing Rhizobium species as w e l l as the plant-transforming Agrobacterium species. The s tudy of s ignal compounds i n relat ion to the b io logy of Agrobacterium tumefaciens is of considerable significance because successful gene m a n i p u l a t i o n i n plants using this microbia l vector is absolutely dependent on these chemicals. A, tumefaciens is a gram negative soi l bacterium which causes crown gall disease of a wide variety of dicotyledonous plants (DeCleen and Deley, 1976). Smith and Townsend (1907) original ly reported the isolation of the bacterium responsible for the disease. Since that time, and particularly recently, a phenomenal amount of research has been conducted on this subject. By the beginning of the 1980's it was established that each virulent strain of A. tumefaciens possesses a large (ca. 200 kb) tumor-inducing plasmid (pTi). A l s o , it was established that the bacterium causes a neoplastic growth of the plant tissue by transferring part of this D N A (the T - D N A ) into the host plant genome (Chilton et al, 1977; Thomashow et al, 1980; Yadav et al, 1980, Chi l ton et al, 1980; Wil lmitzer et al, 1980; Zambryski et al, 1980). N o other procaryotic organism is k n o w n to be capable of this feat of natural genetic engineering, the transformation of eucaryotic cells. The T - D N A includes genes w h i c h encode enzymes of auxin (Schroder et al, 1984; Thomashow et al, 1984) and cytokinin biosynthesis (Akiyoshi et al, 1983; A k i y o s h i et al, 1984), and these genes are expressed in the transformed plant cell (Hille et al, 2 1984a; W i l l m i t z e r et al, 1981). A l s o present i n the T - D N A is a locus confer r ing on the transformed plant cells the ab i l i ty to synthesize characteristic amino acids called opines (reviewed by Nester et al, 1984). Opines produced by the transformed plant cells are catabolized by A. tumefaciens strains which possess specific loci for opine catabolism located on the T i p l a s m i d but not in the T - D N A . Opine product ion confers an advantage on the infecting strain of A. tumefaciens which has the ability to use the opines as a sole carbon and nitrogen source. In addition, it has been f o u n d that the opines stimulate conjugative p T i transfer i n A. tumefaciens (Klapwijk et al, 1978; Petit et al, 1978). It is unclear how such a system evolved, and the exact route by which the transforming D N A reaches the plant genome remains unknown. Useful vectors for genetic engineering i n plants may be constructed by replacing T-D N A genes wi th new genes of interest. Plant cells are then infected w i t h the bacterium containing the modif ied Ti -p lasmid, transformants are selected, and f inal ly plants are regenerated from the transformed cells. This system has been successful i n a number of cases (Goodman et al, 1987; Gaffer and Fraley, 1989, and references therein); however, it should be emphasized that it is ultimately dependent on the ability of the bacterium to detect susceptible cells and express a number of genes essential for successful transformation of the plant genome. It has therefore become of considerable importance that the molecular mechanism by which the bacterium accomplishes this feat be understood i n greater detail. Early studies indicated that attachment of the bacteria to the host plant cells occurs i n a site-specific manner (Lippincott and Lippincott , 1969; Smith and H i n d l e y , 1978). Attachment has been considered an essential stage in crown gall tumorogenesis (Lippincott and Lippincott, 1969). Attachment of A. 3 tumefaciens to the surfaces of a number of plant cell types has been studied by a variety of methods (Gee et al, 1967; Bogers, 1972; Smith and H i n d l e y , 1978; Douglas et al, 1982; Matthysse and Gurl i tz , 1982; Sigee et al, 1982, Draper et al, 1983; Douglas et al, 1985a; Graves et al, 1988). Hasezawa et al (1983) reported the introduction by endocytosis of A. tumefaciens spheroplasts into Vinca rosea protoplasts. A. tumefaciens can attach to suspension culture cells of both monocotyledons and dicotyledons (Matthysse and Gur l i tz , 1982; Douglas et al, 1985a) and can attach either to intact cells or plant protoplasts (Matthysse et al, 1982), but, interestingly, it cannot b ind to cells of carrot suspension cultures which have been induced to form embryos (Matthysse and Gur l i tz , 1982). In general, the evidence f rom several labs suggests that y o u n g , developing cell walls (or damaged cell walls and those under repair at w o u n d sites) are prime targets for binding by the bacteria. Both bacterial and plant components for this b inding have been examined (Lippincott et al, 1977; Lippincott and Lippincott , 1978; Gur l i tz et al, 1987). The plant component appears to be a plant cell w a l l pectin or protein to w h i c h a bacterial lipopolysaccharide binds. A number of these studies show that the bacterial cells attached i n a nonrandom manner; the cells attach to numerous sites on the cell surface first singly (by one end) and later i n clusters. The elaboration of cellulose microfibrils is thought to bind the bacteria f irmly to one another and onto the host cell surface (Matthysse et al, 1981; Matthysse, 1983). In addi t ion , numerous p i l i are produced (Lacey Samuels, unpubl ished observations). Electron microscopy by a number of research groups has revealed that a membranous structure subsequently envelopes the attached bacteria. 4 During the last decade, the genetics of A. tumefaciens, and especially of the early events of the infection process, became much better understood. In a d d i t i o n to the T i p l a s m i d virulence (vir) genes descr ibed b e l o w , chromosomal genes affecting virulence, eg. chvA and chvB (Douglas et al, 1985b) and chvE (Huang et al., 1990), cellulose synthesis (eel) (Mattysse, 1983; Robertson et al, 1988), bacterial attachment (pscA and att) (Thomashow et al, 1987; Robertson et al, 1988) and cellobiase (abg) from Agrobacterium (sp. strain A T C C 21400) have been examined (Wakarchuk et al, 1988). A l l of the nonattaching Agrobacterium mutants reported to date (chvAB, pscA and att mutants) are chromosomal mutants. chvE codes for a protein homologous to periplasmic receptor proteins involved i n chemotaxis and uptake of sugars. chvE mutants display strongly attenuated virulence and restricted host range. Detection of susceptible host cells and early stages of tumorigenesis are mainly controlled by a set of p T i genes k n o w n as the virulence (vir) genes (Garfinkel et al, 1980; Klee et al, 1983; Horsch et al, 1986). Stachel and Nester (1986) investigated the genetic and transcriptional organization of the vir region of Agrobacterium tumefaciens by saturation mutagensis of cloned portions of the vir region w i t h the modif ied transposon T n 3 : : H o H o l . This transposable element contains the 8-galactosidase structural gene (lacZ), and is engineered such that both transcriptional and translational gene fusions may be obtained. W h e n lacZ is inserted i n frame w i t h a vir gene, 13-galactosidase activity can be measured quantitatively to monitor vir gene activation. Reporter gene fusions were used to demonstrate that the vir genes are expressed upon cocultivation of the bacteria w i t h host plant cells (Stachel et al, 1985b; Stachel et al, 1986), and that some diffusible, low molecular weight plant cell factor induced this response. This plant factor is required i n the 5 early stages of tumorigenesis, and therefore is required i n transformation of plant genomes. For these reasons, research has been directed towards (1) revealing the mechanism(s) involved i n vir gene expression, (2) identi fying the vir gene products, and (3) identifying the plant secondary metabolites which may induce or repress vir activity. The genetic and transcriptional organization of the vir region of the w i d e host range A. tumefaciens T i -p lasmid p T i A 6 (Figure 1.1) has been examined by a number of independent research groups. Two of the vir genes (A and G) are regulatory i n nature (Winans et al., 1986; Stachel and Z a m b r y s k i , 1986-a). Apparent ly these act as a two component regulatory system to transmit to the bacterium information about external conditions; they trigger expression of the vir regulon if the external conditions are appropriate. The vir A gene product, the V i r A protein, is also a host range determinant and is thought to be the environmental sensor of the plant der ived inducer molecules (Leroux et al, 1987). The V i r A protein is a transmembrane protein homologous to the sensor component of other two component systems (Winans et al, 1986) and it l ikely interacts w i t h both plant der ived phenolic compounds and monosaccharides (Shimoda et al, 1990). It was recently determined that autophosphorylation of the V i r A protein is required for induction of the vir regulon (Jin et al, 1990a). The activated form of the V i r G protein specifically binds to 5' nontranscribed sequences of the vir genes (Jin et al., 1990b; Pazour and Das, 1990). virC mutants display attenuated virulence or altered host range (Hille et al, 1984b; Hooykaas et al, 1984; Yanofsky et al, 1985; Yanofsky and Nester, 1986). The virD operon is k n o w n to encode a site specific endonuclease which recognizes and cleaves the left and right border sequences of T - D N A (Yanofsky et al, 1986; Stachel et al, 1987). It was subsequently determined that 6 T-DNA A B G C D E Figure 1.1 Genetic and transcriptional organization of the vir region of the T i plasmid pTiA6. Arrows indicate the direction of transcription of vir loci. 7 the endonuclease remains associated with (perhaps covalently bound to) the 5' end of the T - D N A intermediate (Young and Nester, 1988). Perhaps the endonuclease also functions within the plant cell to create a site for insertion into the plant genome. The virB operon encodes 11 different polypeptides (Engstrom et al, 1987), thought to be homologous i n funct ion to pi lus proteins, w h i c h are required for a presumed A. tumefaciens-plant cell conjugation event resulting i n T - D N A transfer (Stachel and Z a m b r y s k i , 1986b). A number of factors are required for vir gene activity. For example, continued expression of virD requires: (1) temperatures below 28 C , (2) acidic p H , (3) plant phenolic compounds (eg. AS) , and (4) sucrose (Alt-M6rbe et al, 1989). Interestingly, z>zr-induction can be enhanced w i t h glycine betaine (Vernade et al, 1988). This natural osmoprotectant apparently accelerates adaptation of the bacteria to the acidic induction medium. Activation of vir gene expression is k n o w n to result i n the production of mult iple , linear, single-stranded T - D N A molecules (T-strands) w i t h i n the bacterium (Stachel et al, 1987). However, this is just one of a few potential intermediates i n T - D N A transmission k n o w n to occur i n A. tumefaciens. C i r c u l a r forms of T - D N A have been reported b y several groups (Koukol ikova-Nico la et al, 1985; Machida et al, 1986; Alt -Moerbe et al, 1986; Yamamoto et al, 1987) and double-stranded cleavages, mediated by the virD gene product, also occur within the T - D N A borders (Veluthambi et al, 1987). Presumably, one or more of these T - D N A molecules are the elements which are transferred to the plant genome. They may be transported as a package along w i t h a vir gene encoded protein(s). The virE2 gene product was recently determined to be a s i n g l e - s t r a n d e d - D N A - b i n d i n g prote in that associates w i t h T - D N A (Gietl et al., 1987; Das 1988; Christie et al, 1988). It 8 was proposed that the VirE2 protein is involved i n the processing of T - D N A and i n T-strand protection during transfer to the plant cell. In summary, the vir region comprises a set of genes that, when expressed, enable A. tumefaciens to detect susceptible plant tissue, that prepare the bacterium for its interaction w i t h the plant cell , and that result in the production of the transforming D N A i n a form ready for transmission to the host. The vir loci were identified by random transposon mutagenesis of A tumefaciens. Insertions i n these loci resulted i n avirulence or altered virulence (Garfinkel and Nester, 1980). Expression of the vir genes was studied before their fuctions were known by inserting a "reporter" gene w i t h easily measurable activity. A s was noted above, Stachel et al. (1985b) prepared a Tn3::/acZ transposon for the random generation of B -galactosidase gene fusions and used it to study gene expression in A. tumefaciens. This system is shown diagrammatically i n Figure 1.2. In a strain carrying a vir ::lacZ gene fusion plasmid, the degree of vir-induct ion can be determined s imply by assaying (3-galactosidase act ivity. Us ing this system it was established that, wi th the exception of the regulatory locus vir A, expression of each of the vir genes can be induced by the presence of certain phenolic compounds (Bolton et al, 1986). The regulation of the vir genes of pTiC58 has been examined with virv.lux gene fusions (Rogowsky et al, 1987). After it was established that cocultivation of the bacteria with the host plant cells resulted i n urr-induction, it was determined that the i n d u c i n g agent must have a molecular weight below 1000 (Stachel et al, 1986). Bolton et al (1986) f o u n d that a mixture of l o w molecular weight phenol ic compounds (Figure 1.3) could be used to induce expression of most of the vir genes. H o w e v e r , quantitative analysis of vir gene induct ion by each 9 pSM-a pSM-b gene fusion plasmids Monitor w r gene induction in pSM strains through /3-gal. assay A348/pSM derivative Figure 1.2 Strategy for analysis of signal compound inducible p T i genes. The modif ied transposon T n 3 : : H o H o l carrying lacZ was inserted at numerous sites i n the vir region on a cloning vehicle and pTi-carrying strains also carrying virv.lacZ gene fusion-containing (pSM) plasmids can be used i n z n ' r - i n d u c t i o n bioassays. 1 0 Figure 1.3. Structures of the seven phenol ic c o m p o u n d s examined for z?ir-induction by Bolton et al. (1986). 1 1 component of the mixture was not reported. Stachel et al. (1985b) identif ied t w o ac t ive s i g n a l c o m p o u n d s , a c e t o s y r i n g o n e (AS) a n d oc-hyroxyacetosyringone (HO-AS) (Figure 1.4), from a transformed tobacco root culture and from leaf discs. In that report a few other related compounds were assayed at one or more concentrations for their pzr-inducing activity. Th is comprised a brief structure-activity study w h i c h y i e l d e d some information about the structural features required to confer activity. A t the concentrations tested, none of the compounds examined by Stachel et al. (1985b) were as active as AS . Of the mixture of seven phenolics examined by Bol ton et al. (1986) only v a n i l l i n (4-hydroxy-3-methoxybenzaldehyde) possesses a methoxy group adjacent to the phenolic h y d r o x y l , and indeed, when they were examined individual ly , only this compound was an effective zn'r-inducer (Spencer and Towers, 1988). The reported activity of a higher molecular weight and apparently proteinaceous inducer of the virC locus (Okker et al, 1984) remains at odds wi th the activity of the lower molecular weight phenolic compounds. Stachel and Zambryski (1986b) have referred to the A. tumefaciens-plant cell interaction as "a novel adaptation of extracellular recognition and D N A conjugation". The presence of the plant cell wa l l and nuclear envelope present significant obstacles for the transfer of D N A . The exact mechanism by w h i c h T - D N A reaches the plant genome from the T i p l a s m i d remains u n k n o w n . It seems that aside from research into vir gene expression, a significant step i n understanding crown gall tumorigenesis w i l l be one of demonstrating the exact sites of bacterial cell attachment, involvement and production of p i l i , and transfer of the T - D N A package across the host cell wal l and membrane. 1 2 Figure 1.4 The structures of acetosyringone (AS) and a -hydroxyacetosyringone ( H O - A S ) . These acetophenone vir-inducers were isolated from transformed tobacco root culture conditioned medium by Stachel et al. (1985b). » 1 3 In the Rhizobiaceae, chemotaxis towards aromatic and hydroaromatic compounds has been demonstrated (Parke et al., 1985; 1987). It has been impl ic i t i n the reports concerning signal compounds for A. tumefaciens, that the bacteria are attracted to susceptible plant tissues by f o l l o w i n g a concentration gradient of the virulence induc ing substances and some support for this idea was obtained by Ashby et al. (1987, 1988). Parke et al. (1987) have s h o w n that A S is not a potent chemoattractant for A. tumefaciens, but that certain other phenolics (gallate, B - r e s o r c y l a t e , protocatechuate, p-hydroxybenzoate, and vani l l in (i.e. a number of the phenolics examined by Bolton et ah, 1986) induce chemotactic behaviour. They showed that chemotaxis towards ufr-inducers is constitutively expressed i n the absence of the Ti-plasmid. However, Ashby et al. (1988) subsequently demonstrated that chemotaxis i n strain C58C 1 is T i plasmid-specified. Another T i p lasmid gene is induced by plant phenolic compounds. The tzs locus, located within the nopaline-type T i plasmid vir region, encodes a d i m e t h y l a l l y l transferase (an enzyme i n cytokinin biosynthesis) whose expression is induced i n a manner similar to that of the other vir loci (John and Amasino , 1988). Induced expression of the gene at this locus by A S was f o u n d to be pH-dependent i n octopine strains and pH- independent i n nopaline strains. Three possible roles (which need not be exclusive) were suggested for this plant-inducible cytokinin production. These are "(i) to condition plant cells to a state in which the transfer of D N A from the bacteria to the plant cell is optimal, (ii) to ensure that plant cells pass through stages of the cell cycle i n which T - D N A integration can occur, or (iii) to stimulate high levels of postintegration T - D N A expression, thus leading to r a p i d tumor development" . 1 4 Perhaps f o l l o w i n g detection of l i g n i n precursors a n d other phenylpropanoid metabolites, the increased levels of cytokinins, resulting from the plant-induced expression of the tzs locus, induces cell divisions and such changes i n the host cell wal l as to expose binding sites to the bacteria. After b inding, a bacterial cellulase may assist i n creating a passageway for T-D N A transfer i n the presumed conjugation-like process. Subsequent to passage of the T - D N A complex into the host cytoplasm, the D N A of a plant cell i n the process of divis ion w o u l d be more easily accessible to T - D N A integration. Prior to m y studies, the newly described compounds A S and H O - A S were generally regarded as unique chemicals which A. tumefaciens detects i n nature and which trigger the initial events within the bacterium, resulting i n tumor formation. Reports have appeared i n the literature concerning the use of w o u n d exudates f rom host plants or of A S to induce virulence of A. tumefaciens and thereby to extend the normal host range (Schafer et al., 1987) or to boost transformation efficiency (Sheikolleslam and Weeks, 1987; Mathews et al., 1990). However, it has not been shown that A S is the signal molecule produced by any susceptible host other than Nicotiana tabacum. Section 1.2.1, below, describes the analysis of structure-activity relationships i n vir gene expression (Paul Spencer, M.Sc. Thesis; Spencer and Towers, 1988). The results of this work suggested other common plant phenolics may be involved in signalling A. tumefaciens, and serves as a point of reference i n the search for novel mr-inducers i n plant-derived exudates. Chapter 2 describes the isolation of a potent uir- inducer c o m p o u n d f r o m grapevine cultivars, which are the hosts for l imited host range ("LHR") A. tumefaciens strains. Chapter 3 describes new methods for, and the results of, a survey of naturally occurring wide host range ("WHR") signal molecules. 1 5 Data therein suggest that commonly occurring plant phenolics other than A S are important signal molecules, and indicate that the natural occurrence of acetophenone signal compounds may be restricted to the Solanaceae. In Chapter 4 an analysis of induced vir gene expression i n the related species A. rhizogenes is presented. Chapter 5 describes the application of combined gas chromatography-mass spectrometry (GC-MS) to the analysis of p i r - i n d u c i n g phenolic mixtures. 1 6 1.2 Signal molecules inducing gene expression i n the Rhizobiaceae 1.2.1 Structure-activity analysis of A. tumefaciens vir gene expression This section describes the activity of a range of phenolic compounds w h i c h can induce vir gene expression i n A. tumefaciens, and was reported i n detail elsewhere (Paul Spencer, M.Sc. Thesis, Spencer and Towers, 1988). The results are summarized here to provide a point of reference i n the search for novel pir-inducers i n plant-derived exudates (Chapters 2 and 3). Analysis of structure-activity relationships of vir gene inducers indicated that a number of k n o w n phytochemicals are l ikely involved i n inducing vir gene expression i n A. tumefaciens (Spencer and Towers, 1988). A s a part of m y Masters Thesis, I reported the m'r-inducing activity over a range of concentrations of a variety of plant-derived and synthetic phenolic compounds w i t h structures related to that of A S , and presented new information regarding the structural features involved i n the activation of vir genes. (3-galactosidase activity was assayed as a measure of vir gene induction i n the A. tumefaciens s t r a i n A348/pSM358 (a C58 derivative harbouring pTiA6) which also carries the virE::lacZ gene fusion plasmid pSM358 (Bolton et al, 1986; Stachel et al, 1985a and b). The activities of some cinnamic and benzoic acid derivatives, chalcones, and of the l ignin precursors sinapyl alcohol and coniferyl alcohol were inc luded. A number of these compounds were chosen for that study because they were k n o w n to be of considerably widespread occurrence, or because they are ubiquitous i n plants, eg. monolignols. Of the mixture of seven phenolic compounds reported by Bolton et al. (1986), catechol, B-resorcylic acid, and gallic acid exhibited very l o w , but detectable levels of i n d u c t i o n ; protocatechuic , p y r o g a l l i c , and p-1 7 hydroxybenzoic acids d i d not induce vir expression signif icantly above background levels. Only vani l l in greatly induced the expression of vir genes. Apparent ly , at least one r ing methoxy substituent next to a para hydroxy group is required for activity. Consistent wi th this observation, Stachel et al. (1985b) repor ted negl ig ib le i? i r - induct ion i n response to 50 U.M p-hydroxyacetophenone. Further analysis was necessary i n order to determine what other structural features (what other r ing substitutions) conferred activity. The structures of sixteen urr - inducing phenolic compounds were examined for the purpose of obtaining an understanding of the structure-activity relationships of pz'r-induction. These compounds may be assembled into four groups (Figure 1.5): 1. acetophenones and benzoic acid derivatives, 2. monolignols , 3. cinnamic acid derivatives, and 4. chalcone derivatives. Each structure contains a guaiacyl or syringyl nucleus, most possess a carbonyl g r o u p , a n d m a n y are c o m m o n p l a n t - d e r i v e d c o m p o u n d s of the p h e n y l p r o p a n o i d pathway (Harborne, 1989). The pir -act ivat ion curves obtained for a number of these compounds are shown i n Figures 1.6 and are discussed briefly below. The l o w e r ac t iv i ty of 4 -hydroxy-3 -methoxyacetophenone , or "acetovanil lone" (of guaiacyl substitution) i n comparison w i t h that of acetosyringone (of syringyl substitution) indicates that for acetophenones a syr ingyl nucleus is more effective at zn'r-induction than is a guaiacyl nucleus. This supports the results of Stachel et al. (1985b) who assayed the relative activities of these two acetophenones at four concentrations using a virB::lacZ strain of A. tumefaciens. They suggested that the acetyl substituent is important for activity because, in comparison, the carboxylic analogue of A S , syringic acid, was less active. I found that the methyl esters were roughly as 1 8 C H 3 0 2. C H , 0 C H 3 0 C H 2 O H O x ^Ri O H O C H , O H l a H b H c C H 3 d C H 3 e O H R 2 H O M e v a n i l l i n s y r i n g a l d e h y d e H acetovanillone O M e a c e t o s y r i n g o n e OMe syringic acid f OMe OMe Me-syringate 2a R= H Coniferyl alcohol b R= OMe Sinapyl alcohol R i R 2 3 a H H v a n i l l a l a c e t o n e b O H H ferulic acid c O H H sinapinic acid d O M e H Me-ferulate e O M e O H 5-OH-Me-ferulate f O M e O M e Me-sinapinate Chalcones 4a R= H 2* ,4 \4- (OH) 3 -3-OMe b R= OMe 2' ,4\4-(OH) 3 -3,5-(OMe) 2 O H O Figure 1.5 The 16 chemicals used i n the study of structure-activity relationships, arranged into 4 classes. 1 9 1000 1 0 " 2 1 0 " 1 10° 1 0 1 1 0 2 10 Concentration (uM) Figure 1.6 virE expression i n A. tumefaciens i n response to phenol ic compounds. F o l l o w i n g 10 hrs. incubation w i t h a compound i n aqueous solut ion, B-galactosidase activity was assayed i n the strain Agrobacterium A348/pSM358, which carries a virEv.lacZ reporter gene fusion plasmid (data from Spencer and Towers, 1988). 20 active again as the acetophenones, suggesting that the acetyl group can be replaced by an ester group. The curves of activity induced by the chalcones are somewhat different than those of any of the other compounds examined. 2',4',4-Trihydroxy-3-methoxy-chalcone displayed its greatest vir - inducing activity at 10 | i M . The m a x i m u m levels of induct ion by a l l of the other compounds (except syringaldehyde) were obtained at the highest concentration tested, 200 u M . Unl ike any of the other compounds, this chalcone was capable of low level tnr- induction at 0.1 | i M . The curve for 2' ,4' ,4-trihydroxy-3,5-dimethoxy-chalcone is shifted more to the right than the other chalcone, closer to that of acetosyringone, and its maximum activity is observed at 50 u M . Perhaps this is as a result of the syringyl substitution of its B-ring affording a structure more similar to that of acetosyringone. Neither of these chalcones is very soluble i n the buffer used, but they do exhibit significantly greater activity at lower concentrations than do any of the simpler phenolics tested. A number of chalcones are k n o w n to exhibit biological activity (Dhar, 1981) and it w i l l be interesting to determine whether chalcones act as naturally occurring zn'r-inducers. Coinc identa l ly , a new chalcone from alfalfa (4,4'-dihydroxy-2'-methoxychalcone) was recently found to induce nodulat ion (nod) gene expression i n Rhizobium meliloti ( M a x w e l l et al, 1989; see section 1.2.2). Previously, this chalcone had been reported as a stress metabolite from Pisum sativum (Carlson and D o l p h i n , 1982). nod-inducers of Rhizobium species are discussed below, i n section 1.2.2. A t this point, I emphasize that Maxwell 's report confirms that chalcones play a role i n s ignal l ing between plants and microbes. A l s o , chalcone-mediated gene activation i n these related genera is demonstrative of a close relationship i n signalling systems between agrobacteria and rhizobia. 2 1 Other compounds tested, most of which possessed guaiacyl or syringyl substitution patterns but which exhibited little or no tn'r-inducing activity, i n c l u d e the f o l l o w i n g : p h l o r i d z i n , c h r y s o s p l e n o l - 6 - C - g l u c o s i d e , homoeridiodictyol , tr icin, 3,5,7,4'-tetrahydroxy-3'-methoxy f lavonol , plicatic a c i d , c o n i d e n d r o n , substi tuted aurones, v a n i l l o y l m e t h y l ketone, 5-hydroxyvani l l in , dihydrodiferulic acid, 5-hydroxyferulic acid, isoferulic acid, gluco-ferulaldehyde, and coniferin. M o r r i s and M o r r i s (1990) reported coniferin to be an active signal compound obtained f rom Pseudotsuga menzesii stem tissue. In this case, the Agrobacterium strain used also exhibited glucosidase activity and it is l ikely that i n this case the bacteria generated the active aglycone coniferyl alcohol from the glucoside. A p p a r e n t l y , 5-hydroxylat ion of any active compound possessing a guaiacyl nucleus decreases the compound's activity. Thus whereas ferulic acid is somewhat active, 5-hydroxyferulic acid is inactive. This decrease i n activity appears to be true even of 5-hydroxy methyl ferulate, w h i c h retains activity similar to that of methyl ferulate, but reaches its m a x i m u m at a concentration of 50 j i M . The inactivity of 5-hydroxyferulic acid is of interest because it occurs ester-bound i n the cell walls of grasses (Ohashi et al, 1987). Perhaps this is a factor i n the resistance of certain monocots to infection by A. tumefaciens. Repression of the expression of vir genes is another important subject requir ing research, as it too may be an important factor i n host range determinat ion. A l t h o u g h i n h i b i t i o n of vir - induct ion by a phenol ic compound was not demonstrated during the course of the structure-activity analysis/certain observations lead one to speculate about the possibility that phenolic m'r-inhibitors exist which are common to all monocots. This w o u l d expla in w h y the monocots are natural ly resistant to infection by A. tumefaciens, and also w h y preincubation of the bacteria w i t h a k n o w n inducer (Schafer et al, 1987) can then allow transformation. In fact, it has n o w been established that inhibitory compounds are released from wounded Zea mays tissues (Sahi et al, 1990). In this case, the wel l k n o w n hydroxamic acid D I M B O A and related compounds were found to inhibit growth and virulence of A. tumefaciens. Identif ication of D I M B O A and related compounds by H P L C is reported i n Chapter 3. The structures of the aglycones of gluco-ferulaldehyde and coniferin meet the putative requirements of an active signal compound (sumarized below) and yet the glycosides were found to be inactive. A l though only two phenolic glycosides were tested, the results indicate that, dur ing the exposure time of these assays, Agrobacterium vir genes are not induced by such compounds . If the vacuolar phenolics, w h i c h must be exuded u p o n w o u n d i n g , are a source of pir - inducing phytochemical precursors, then it appears that plant glucosidases must act to yie ld the effective compound. Results presented i n Chapter 3 suggest that glucosidases do indeed act on phenolic glucosides upon wounding. Previously, I suggested that the role of glycosides such as coniferin [4-(3-hydroxy-l-propenyl)-2-methoxyphenyl-D-glucopyranoside] and isoconiferin [ l-(4-hydroxy-3-methoxyphenyl)-propenyl-3-D-glucopyranoside] should be investigated (Paul Spencer, M.Sc . Thesis; Spencer and Towers , 1988). Coinc identa l ly , coniferin is found i n Asparagus (Merck Index, 10th edn. (1983) Merck Rahway), which appears to be one of the few monocots f rom w h i c h Agrobacterium-transformed tissue has been obtained by standard methods (Hernalsteens et al, 1984). A s noted above, for a certain A. tumefaciens strain, coniferin was recently reported to be the active compound 23 present i n conifer extracts (Morris and Morr is , 1990). Certain sugar esters of uir-inducing phenolics are also effective uir-inducers (see Chapter 3). It is interesting to note that Agrobacterium species are k n o w n to degrade the l ignin model compounds a-conidendron and veratrylglycerol-B-coniferyl ether (Subba Rao et al, 1971). In this study, A. tumefaciens vir genes were not activated by conidendron. However , this brings up an important point , namely, that A. tumefaciens may chemically alter the compounds w h i c h induce virulence. Tracer studies should be conducted i n order to determine whether this is indeed the case, and the metabolites should be identif ied. In addi t ion to the inactive compounds listed above, most of the common phenolic compounds used by Bolton et al. (1986) were inactive. O n l y v a n i l l i n caused any significant u/r-induction. V a n i l l i n is not a l ignin precursor, although it is a breakdown product from l ignin (Freudenberg and N e i s h , 1968), as is syringaldehyde. This may indicate that both l i g n i n precursors and l i g n i n degradation products function i n nature as vir-inducers , w h i c h i n turn suggests that A. tumefaciens may be generally attuned to l ignin metabolites. Interestingly, i n comparison w i t h the response i n d u c e d by v a n i l l i n , the remaining compounds (gallic, (3-resorcylic , pyrogal l ic , p-hydroxybenzoic, and protocatechuic acids, and catechol) were essentially inactive. None of these inactive compounds possesses a guaiacyl or syr ingyl nucleus, which explains their lack of activity. It remains to be determined i n what manner each of these compounds effects the activity of v a n i l l i n . In summary, two basic structural features together are required to confer activity upon a compound. W i t h the exception of the monolignols and the chalcones, these features are: 1) guaiacyl or (conferring enhanced activity) syringyl substitution on a benzene ring, and wi th the exception of the monol ignols , 2) a carbonyl group on a substituent para to the hydroxy substituent on the ring. There are restrictions on the nature of the carbonyl carbon. It may be one or three carbon atoms removed f rom the r ing . However , to confer maximal activity, i n the latter case there must be a double bond between the carbonyl carbon and the r ing, as is present i n the chalcones and cinnamic acid derivatives. Furthermore, the carbonyl group of a free acid is less effective than that of the corresponding ester. The methyl esters of each of ferulic, syringic, and sinapic acids exhibited significantly greater activity than the corresponding free acids. Coincidentally, one of these esters, methylsyringate, was isolated from grapevine cultivars (described i n Chapter 2). Perhaps the activity of esters of phenolic acids could be used to explain the early result (Okker et al, 1984) w h i c h indicated the involvement of a proteinaceous inducer. The inducer obtained may have been a protein ester of some phenolic acid. Esterification alters the solubility of the compound. In addition, esterification prevents one oxygen of the carboxyl group from forming a partial double bond, thereby rendering the carbonyl group more reactive. In these cases, and the case of the aldehydes and chalcones, this carbonyl group forms the terminus of a conjugated double bond system running from the h y d r o x y l group and through the ring. Lastly, flavonoids other than the chalcones were inactive. Cel l -ce l l s ignal l ing must occur naturally i n media of considerable chemical complexity, w i t h both inducers and inhibitors present. Therefore, successful induct ion vir genes w o u l d require the correct balance between inducer and inhibitor molecules. These factors may be used to explain the artificial extension of the host range of A. tumefaciens (Schafer et al, 1987), as 25 wel l as the increased transformation efficiency obtained by pre-induction of A. tumefaciens w i t h induc ing exudates or A S (Sheikolleslam and Weeks, 1987). A number of a i r - inducing phenolic compounds are of widespread occurrence amongst dicotyledonous plants. The l ignin precursors coniferyl and sinapyl alcohol must be ubiquitous amongst susceptible hosts. It w o u l d therefore be possible to conclude that, if present in the correct concentration, the presence of one or another of these compounds alone w o u l d determine whether a given plant is susceptible to infection by Agrobacterium. However , it is w e l l k n o w n that monocots also produce phenolic compounds, even exuding phenolic acids such as syringic, sinapic and ferulic acid into the rhizosphere from intact roots (Tang and Young, 1982), and yet, w i t h few exceptions (Hernalsteens et al, 1984), monocots lie outside of the natural host range of any strain of Agrobacterium. Concentration effects, as wel l as the action of inhibitors of a i r - induct ion , may be factors i n the resistance of monocots to crown gall tumorigenesis. Restrictions i n host range remain a significant problem i n the use of this organism as a vector for genetic engineering in plants. Factors other than inducer/ inhibi tor phytochemistry may wel l be involved i n the immuni ty of monocots to transformation by Agrobacterium. U s i n g the results of the structure-activity analysis as the foundation for subsequent research, this thesis reports the isolation and identification of phenolics affecting virulence f rom both monocotyledons and dicotyledons. Some methods and results presented i n this thesis may facilitate research concerning factors determining host range and susceptibility to crown gall disease. Important and unanswered questions concerning znr-inducers of A. tumefaciens are: (1) w h i c h phenolic compounds are natural ly occurr ing 26 signal molecules, and (2) what are their immediate precursors i n the unwounded plant? In section 1.3 a brief consideration of the possible sources of signal compounds from wounded plant cells is presented. Some answers to the question of biosynthetic precursors of Agrobacterium s ignal compounds are described i n Chapter 3. 1.2.2 F lavonoid nod gene inducers of Rhizobium species Comparisons may be made between the chemistry of Agrobacterium virulence- induct ion w i t h that of nodulat ion (nod) gene induct ion i n the closely related genus Rhizobium. The nod genes control early events i n the formation of nitrogen f ix ing nodules on the roots of leguminous plants. Express ion of the nod genes by Rhizobium species is essential for the successful nodulat ion of host plant species, and this gene expression is induced by host plant-derived flavonoids. The activity of the more typical f lavonoids represents a significant difference i n the range of structures i n d u c i n g gene expression i n the two genera. The structure-act ivity relationships reported i n this thesis are clearly different from those reported for the activation of nod genes in Rhizobium species (Peters and L o n g , 1987; Zaat et al, 1989). H y d r o x y l a t e d flavones, isoflavones, d ihydrof lavonols , f lavanones, and even a chalcone, i n n M to | i M concentrations induce expression of nod genes (Peters et al, 1986; Redmond et al, 1986; F i rmin et al, 1986; Sadowsky et al, 1988; Maxwel l , 1989; Zaat et al, 1989). The compounds i n v o l v e d i n nod gene induct ion include the w e l l k n o w n f lavonoids luteo l in , apigenin, inteol in , nar ingenin , e r iodic tyo l , geraldone, and daidzein. The structures of a number of the active flavonoids which have been identified from host plants are shown in Figure 1.7. F i rmin 27 H O O H Rhizobium trifolii Flavones a. R=H, 7,4'-dihydroxyflavone (DHF) b. R=OMe, 7,4-dihydroxy-3 '-methoxyflavone (Geraldone) Rhizobium fredii Isoflavone (Daidzein) 4',7-dihydroxyisoflavone Rhizobium meliloti Flavanone (weak inducer) 4' ,7-dihydroxyflavanone Flavone (moderate inducer) 4' ,7-dihydroxyflavone Chalcone (strong inducer) 4,4'-dihydroxy-2'-methoxy chalcone Figure 1.7 The structures nod- induc ing f lavonoids recently identified from host plants. 2 8 et al. (1986) d i d not isolate nod-inducing flavonoids from host exudates, but several commercially available f lavonoids were examined (Figure 1.8) i n order to outline the range of compounds which could induce nod gene expression i n R. leguminosarum. Zaat et al. (1989) characterized 7 inducers for the nodA promoter of R. leguminosarum biovar viciae f rom the root exudate of Vicia sativa, and examined structure-activity correlations through the application of 34 authentic standard flavonoids. Most Rhizobium species are relatively specific for their respective host plant species, and each Rhizobium species appears to exhibit a relatively high degree of specificity towards flavonoids from its host. A l s o , the compound w h i c h most strongly induces one species of Rhizobium can act as a potent inhibitor of nod-induction i n another species of Rhizobium. By contrast, the original strain of A. tumefaciens from which the strain used i n this study was derived exhibits a wide host range (WHR), and a comparatively much lower degree of signal compound specificity. F i r m i n et al. (1986) also described a novel phenomenon: some of the very compounds which induce vir genes i n A. tumefaciens, inc luding A S , s t rongly inh ib i t nod gene activation by these f lavonoids . A t higher concentrations most of the tnr- inducing phenolics are bacteriostatic even against Agrobacterium (data not shown), and presumably they act i n this way against Rhizobium species, or they may act more directly by competitive inhibit ion of nod-induct ion. 2 9 Flavones a. Rj=H, R 2 =OH, R 3 =H (Apigenin) b. Ri=OH, R 2 =OH, R 3 =H (Inteolin) c. R ^ O H , R 2 =H, R 3 =H d. .R!=H, R 2 =OH, R3=glc (Apigenin-7-O-glucoside) Flavanones a. R ^ H , R 2 =OH (Naringenin) b. R ^ O H , R 2=OMe (Hesperitin) c. R ^ O H , R 2 =OH (Eriodictyol) Figure 1.8 Authentic flavonoids used i n the analysis of R. leguminosarum nod-induction by F i r m i n et al. (1986). 30 1.3 Phenolic biochemistry of wounded plant tissues i n relation to vir gene  expression In the course of their l ives, many plants experience and overcome naturally inflicted wounds. They are surprisingly resilient, capable of healing reactions that are often successful processes. C r o w n gall disease requires of both pathogen and host a coordinated series of biochemical events, including wound-heal ing reactions by the host. The bacterial pathogen, Agrobacteium tumefaciens, causes tumors fo l lowing infection at w o u n d sites on a wide variety of host plants. This process is dependent on the bacteria sensing susceptible host cells by responding to the presence of signal compounds exuded specifically by wounded cells. Such compounds are also produced by suspension culture cells, presumably because the culture conditions mimic the w o u n d e d state. Signal compounds are rapidly elaborated by newly w o u n d e d tissues, but the source(s) of these interesting compounds has not been established. This section reviews informat ion regarding w o u n d biochemistry as it may relate to infection by A. tumefaciens. The recent discoveries concerning s i g n a l l i n g i n p lant -microbe interactions have opened up new areas of research i n natural products chemistry. Important roles are being established for some classes of plant secondary metabolites, the functions of which, unti l now, were unknown. A s described above, the virulence (vir) genes of this organism are switched on when it encounters plant phenolic compounds of guaiacyl or syr ingyl r ing substitution (Stachel et al, 1985; Spencer and Towers, 1988; Spencer et al, 1990). The k n o w n Agrobacterium s i g n a l c o m p o u n d s i n c l u d e the acetophenones A S and H O - A S (Stachel et al, 1985), and the methyl ester of 3 1 syringic acid (Chapter 2, Spencer et al, 1990). However, the range of effective phenolics may not be l imited to C6-C2 and C6-C1 compounds. A number of cinnamic acid derivatives (phenylpropanoids), i.e. C6-C3 compounds, are also active (Spencer and Towers, 1988). The l ign in precursors coniferyl and sinapyl alcohol, ferulic and sinapic acids and some of their esters are active in vitro, and may be naturally occurring signal compounds (described i n Chapter 3 ) . U n t i l the completion of this thesis, however, these C6-C3 compounds had not been isolated from wounded plants on the basis of their biological activity. Whereas a number of c o m m o n l y o c c u r r i n g , potent ia l s igna l c o m p o u n d s are k n o w n , only three p r e v i o u s l y u n k n o w n compounds mentioned above have been identif ied f rom w o u n d e d plant tissues. A growing number of research papers are appearing i n the literature concerning the effects of these plant phenolics on A. tumefaciens. However , few of these reports have considered the biosynthetic source(s) of the inducers of virulence. It has been suggested by some authors that the acetophenones encountered by A. tumefaciens are intermediates in the biosynthesis of l ignin (eg. Stachel et al, 1985b). This seems to be an unrealistic v i e w when one considers that only C6 -C3 compounds have been identif ied as monomeric precursors or components of the l ignin polymers. Certain cell w a l l bound phenolics may be liberated upon wounding or in connection w i t h cell w a l l - repair. These and phenolic glucosides appear to be more l ikely sources of signal compounds. In this section I shall review information available on the natura l occurrence and possible routes for the p r o d u c t i o n of s ignal compounds. Extracts from fresh, nonwounded plant tissues apparently contain little or no u/r - inducing substances (Stachel et al, 1985). It was also shown that 3 2 cycloheximide treated w o u n d e d tissues also fai led to produce the vir-inducers . This suggests that w o u n d - i n d u c e d prote in synthesis and subsequent enzymatic activity are required for biosynthesis of these phenolics. The effects of cycloheximide on the biosynthesis of signal molecules are mentioned i n sections 1.3.5 and 1.3.6, and some experimental results are described i n Chapter 3. Since it can be assumed that the plant does not synthesize such compounds solely for the purpose of signalling a pathogen, other reasons for their existence (in planta roles) must be found. So far, none has been established. Is it possible that these plant secondary compounds solely represent "metabolic chatter", w h i c h i n Agrobacterium plays such an important role? Knowledge of the source of these compounds might aid our understanding of the raison d'etre of signal molecules. The sources of these compounds might include de novo biosynthesis v ia the phenylpropanoid pathway, synthesis from vacuolar precursors (eg. glycosides), liberation of cell wal l -bound phenolics, including those of l ignin or suberin (or precursors i n the wound-induced biosynthesis thereof). Wound-heal ing reactions have been the subject of microanatomical and biochemical analyses. Various biochemical events occur rapidly upon • tissue damage. Cel l contents are mixed, react w i t h one another, and are exuded from the wound site. In addition, marked changes are induced i n the secondary metabolism of neighboring cells. Infection by microorganisms can stimulate the production of plant defence compounds called phytoalexins, but this is not considered at length in this thesis. Indeed, this may occur when plants are inoculated w i t h Agrobacterium. Perhaps phytoalexins are i n v o l v e d i n the observed hypersensitive response of certain grapevine cultivars to incompatible Agrobacterium strains (Yanofsky et al., 1985). D u r i n g the first week fol lowing wounding , some cell divisions occur and a layer of plant cells differentiates into w o u n d periderm (Juniper and Jeffree, 1983). Lignification and suberization of these cells serves to w a l l off the injured area, preventing dry ing and infection. However , this is l ike ly beyond the w i n d o w of opportunity for infection by Agrobacterium. A number of subjects relating to the origin and nature of signal compounds are considered below. 1.3.1 Phytoalexins A n early definition of phytoalexins states that: "These are compounds (nonspecific toxins or antibiotics) which can inhibit the development of the pathogen but are only formed or activated when the latter comes into contact w i t h the host cells" (Swain, 1977). A more contemporary def ini t ion of phytoalexins includes those compounds synthesized i n response to infection or challenge of plant tissues wi th some pathogen or elicitor, for example a fungal cell wal l fraction (reviewed by Friend, 1985). In the generally accepted sense of the term, therefore, the definition of phytoalexins is not sufficiently broad to include plant metabolites w h i c h act as A. tumefaciens s ignal compounds. Phytoalexins may be present at low levels i n uninfected tissues, but they can accumulate to very high levels fol lowing elicitation. I found this to be true of acetophenones i n the Solanaceae (described i n Chapter 3). It is quite possible that host plants, or more l ike ly non-host plants, w h e n inoculated w i t h A. tumefaciens, react by producing antibacterial compounds (anti-Agrobacterium phytoalexins). Perhaps this is a factor i n A. tumefaciens host range phenomena, wherein a successful relationship is established only i n those cases in which the bacteria do not elicit a response by the plant. To my knowledge this subject has not been investigated. Such research could be 34 important i n reveal ing w h y the monocots, i n general, are resistant to infection by A. tumefaciens, or w h y grapevine cultivars are susceptible mainly to L H R strains. It is w e l l established that signal compounds are synthesized solely i n response to w o u n d i n g of plant tissues, and that this does not require elicitation by any additional factors. I note that phytoalexins now include stress-related compounds, for example, compounds produced i n response to w o u n d i n g (eg. Basha et al, 1990). A recent report (Threlfall and Whitehead, 1988), which focussed mainly on sesquiterpenoid phytoalexin metabolism i n tobacco cultures, has complicated this issue. These workers studied the el icitat ion of anti fungal sesquiterpenes i n tobacco suspension cultures. Pur i f i ed cellulase not only stimulated sequiterpenoid biosynthesis i n these cultures, but apparently also the accumulation of acetosyringone. The cellulase used was found not to possess any 8-glucosidase activity, so the acetosyringone was not s imply a hydrolysis product of the corresponding glucoside. Thre l fa l l and Whitehead (1988) f o u n d that A S d i d not exhibit antifungal activity against their test organism, so the production of A S does not appear to represent a chemical defence against fungal infection. A l s o , i n comparison w i t h catechol, gallic, pyrogall ic , or protocatechuic acids, A S is only w e a k l y bacteriostatic even at relatively high concentrations (ca. 1 mM )(Paul Spencer, M.Sc. Thesis). In fact, to date no strong antimicrobial activity has been reported for A S , and one must conclude that A S is not a phytoalexin. Threlfall and Whiteheads' results are discussed further under cell wal l -bound phenolics (section 1.3.2). 1.3.2 C e l l wall -bound phenolics Ester and ether-linkages of p-coumaric, ferulic, and dimeric acids to the cellulose or hemicellulose component of cell w a l l polysaccharides is of fairly widespread occurrence (El-Basyouni and Towers, 1964; Harr is and Hart ley, 1976; Hartley and Ford, 1989). These studies have examined graminaceous cel l w a l l phenolics , but such linkages l ike ly occur i n the cel l w a l l of dicotyledons as well . 0 Swain (1977) suggested that the presence of these compounds could be part of a primit ive defence mechanism against attack by pathogens. These esterified phenolics also could act as anchors, or starting points for the synthesis of l ignin or suberin, a means of crosslinking (Figure 1.9) between nonaromatic cell wal l material (Markwalder and N e u k o m , 1976; Iyama et al, 1990), or may limit binding of pathogens to cell walls (Hartley and Ford, 1989). This last idea is probably not a factor i n the resistance of monocots to transformation by A. tumefaciens, because a number of studies have proven that b inding of A. tumefaciens to various monocot cells occurs i n a manner similar to that observed wi th dicot cells (Douglas et al, 1985; Graves et al, 1988). A t least i n certain cases, p-coumaric and ferulic acids are liberated from the cell walls during decay (Karunen and Kalviainen, 1988). Plate assays w i t h cell wal l feruloyl sugar esters (arabinose, xylose and glucose esters) indicate that these compounds are effective vir -inducers (see Chapter 3). Cellulase activity is known to cause the release of water soluble O-[5-0-(rrans-feruloyl)-a-L-arabinofuranosyl]-(l-3)-O-B-D-xylopyranosyl-(l-4)-D-xylanopyranose, or F A X X (Figure 1.10) (Hartley and Ford, 1989). It w i l l be interesting to determine whether under any natural circumstances such cell w a l l fragments such as F A X X influence virulence of A. tumefaciens . 3 6 Bound ferulate Bound ferulate Figure 1.9 Formation of bound diferulate i n cell walls. R=cell wa l l polymer. 3 7 O H Figure 1.10 Chemical structure of F A X X . 3 8 A s noted above, Threl fa l l and Whitehead (1988) reported that acetosyringone is produced i n tobacco cell cultures fol lowing treatment wi th cellulase. It is not k o w n whether the compound is liberated from the cell w a l l or whether it is newly synthesized. The cellulase treatment may mimic the effects of w o u n d i n g , and stimulate de novo biosynthesis of A S , not s imply its release f rom cell walls . However there are data to suggest that compounds other than phenylpropanoids are important constituents of cell wal l s . Hart ley and Ford (1989) noted the relationship between phenolic constituents of cell walls liberated by treatment with alkali (eg. 0.1 M N a O H , 20 °C) and biodegradability wi th cellulase. p-Hydroxybenzaldehyde, vani l l in , syringaldehyde, p-coumaric acid, ferulic acid and dehydrodiferulic acid are released from graminaceous cell walls by m i l d alkali or cellulase treatment. The aldehydes are released as water soluble carbohydrate-aromatic compounds f r o m w h i c h the aromatics are released by further a lka l i treatment. This suggests that the aldehydes are ether l inked to the cell w a l l polysaccharides. The ability of cellulase to release az'r-inducers from cell w a l l material has not been established. It is known that treatment of certain plant tissues w i t h cellulase p r i o r to inoculat ion w i t h Agrobacterium can permit transfomation of species that otherwise resist infection. In Chapter 3, the effect of cellulase on the production of ufr-activating compounds is described. 1.3.3 Lignif ication Unfortunately, many of the studies related to the production of l ignin are based on plant responses to infection and not s imply to w o u n d i n g . H o w e v e r , phenylpropanoid biosynthesis l ikely related to l ignif icat ion has been studied i n potato tuber slices. A n important enzyme, cinnamic acid 4-hydroxylase is strongly stimulated i n aging disks of potato, and roots and tubers of other species (Rhodes and Wooltorton, 1978). Increased peroxidase activities i n sweet potato slices may be related to l ign in biosynthesis since these enzymes are probably i n v o l v e d i n oxidative p o l y m e r i z a t i o n of phenylpropanoid units (the monolignols) leading to l ignin. Spencer and Towers (1988) have shown the l ignin precursors coniferyl alcohol and sinapyl alcohol to be relatively strong az'r-inducers, but so far these compounds have not been detected i n exudates from w o u n d e d plant material as vir- inducing chemicals (see, however, Chapter 3- Taxus baccata). The process of l ignif icat ion is thought to require the phenylpropanoid alcohols (Freudenberg and Neish , 1968). These alcohols must be der ived f rom the corresponding phenylpropanoid acids. In a detailed review of phenylpropanopid metabolism i n cell walls, Yamamoto et al. (1989) discussed the biosynthesis of l i g n i n and the turnover of monol ignols (and their glucosides). Monol igno ls seldom accumulate i n w o o d y tissue, but are incorporated completely into the l ign in framework. In angiosperms the accumulation of E-monolignol glucosides appear to be mainly restricted to the Magnoliaceae and Oleaceae families. In the bark of the American beech (Fagus grandifolia) only the Z-monolignols and their glucosides are present (Lewis et al, 1988). It is thought that the E-monolignols may be used exclusively for l ignin formation. Cel l wal l B-glucosidases are thought to act on the monolignol glucosides to yield the aglycones. The efficient turnover of the aglycones, perhaps i n v o l v i n g enzyme-bound intermediates, may preclude their presence i n w o u n d exudates and detection by A. tumefaciens. Spencer and Towers (1988) reported that the monolignol glucosides were inactive, but suggested that they were a logical precursor of coniferyl alcohol f o l l o w i n g w o u n d i n g and subsequent hydrolysis . The monolignols were 40 included i n the structure-activity study because they possess a propenol side chain, and this permitted comparison wi th the activity of the propenoic acid side chain of the corresponding posit ion 3, 4, and 5-substituted rings. H o w e v e r , they were chosen pr imar i ly because as l ign in precursors they w o u l d be common to all susceptible hosts. L i g n i n composit ion has been studied by analysis of degradation products (reviewed by Gross, 1979). Nitrobenzene oxidat ion yields p-hydroxybenzaldehyde, vani l l in and syringaldehyde, the latter two of w h i c h happen to be potent tnr-inducers. Quantit ies of s y r i n g y l residues i n herbaceous dicotyledon lignins ranges from 10-65%. Lignins of the Poaceae are characterized by a higher proportion of p-coumaryl residues. Perhaps this is a factor in the resistance of monocots to crown gall disease. A l s o , Gross also pointed out that l ignin composition varies greatly between different tissues. Perhaps the c r o w n region of plants are preferentially infected by A. tumefaciens partly on the basis of l ignin composition. I have assayed the effect of p-coumaric acid and found that it had little suppressive effect on vir-induction by acetosyringone. It is conceivable that signal compounds could be derived from l ignin , fo l lowing wounding , by enzymatic activity liberating C6-C2 or C6-C1 moieties. To m y knowledge, no studies have focussed on the l iberation, through natural means, of such low molecular weight units from l ignin. In Chapter 3, an experiment is described i n which the effect of cellulase on the production of acetosyringone from n o n - l i v i n g , solvent extracted, tobacco cell w a l l material was examined. 4 1 1.3.4 Suberization Wound- induced suberin is a complex, polymeric substance produced by wounded tissues to form a barrier to infection by disease organisms (Figure 1.11). Over the last two decades, Kolattukudy has contributed greatly to our knowledge of the composit ion of suberin. The aliphatic components of t h e s u b e r i n s tructure have been p a r t i c u l a r l y w e l l c h a r a c t e r i z e d . Unfortunately, the phenolic components are less w e l l k n o w n than are the aliphatic components, p-coumaroyl and feruloyl residues are thought to be the main phenolic residues to be found i n suberin. Al though pure suberin is impossible to prepare, suberin-enriched extracts can be prepared through treatment w i t h cellulases and pectinases. M o d e r n 1 3 C - N M R techniques have permitted analysis of suberin without extensive sample purification (Garbow et al., 1989). This N M R analysis verified that suberin has phenylpropanoid groups characteristic of l ignin. Depolymerization of suberin involves cleavage of ester bonds. This is accomplished by hydrogenolysis with L i A l H , hydrolysis with alcoholic alkal i , or t ranses ter i f i ca t ion w i t h M e O H a n d BF3 catalyst or N a O M e . Transesterification gives rise to four classes of monomers: fatty acid methyl esters, dicarboxylic acid methyl esters, fatty alcohols, and co-hydroxy acid methyl esters. Combined G C - M S has been used extensively to characterize these components. The phenolic fraction derived from suberin can also be examined by G C - M S . Hydrogenolysates of suberin samples from potato, sweet potato, turnip, rutabaga, carrot, and red beet gave dihydroconiferyl alcohol. Riley and Kolat tukudy (1975) found ferulic and /7-coumaric acids covalently attached to suberin. Apparently no systematic investigation of the phenolic components of suberin has been reported. Figure 1.11 Generalized structure of suberin (after Kolattukudy, 1984). A l l the experimental evidence available on the biosynthesis of the phenolic components strongly suggests that d u r i n g w o u n d heal ing the phenolic components are formed prior to the formation of the aliphatic components. It is noteworthy that, just as washing of explants can inhibit tumor format ion by A. tumefaciens, thorough washing of potato discs immediately after preparation and up to three days fo l lowing preparation severely inhibits suberization. In addit ion to the presence of various phenolics (Chapter 3), G C - M S analysis of virulence inducing mixtures from wounded plant tissue revealed the presence of a number of fatty acids (see Chapter 4). Perhaps these acids are indicators of the process of suberization i n the wounded plant tissue. 1.3.5 De novo biosynthesis of phenolic compounds The synthesis of new phenylpropanoid units upon w o u n d i n g and the induct ion of some of the required enzymes has been studied (reviewed by Rhodes and Wooltorton, 1978; Rhodes, 1985). M a n y of these studies have focussed on the production of chlorogenic acid or scopoletin i n aging potato tuber discs. In carrot tissues, i n addition to stimulation of chlorogenic acid and scopolet in biosynthesis , w o u n d i n g results i n the p r o d u c t i o n of i socoumar ins and chromones (Figure 1.12). 6 - H y d r o x y m e l l e i n , 6-m e t h o x y m e l l e i n , 5 - h y d r o x y - 7 - m e t h o x y - 2 - m e t h y l c h r o m o n e a n d 5,7-d i h y d r o x y - 2 - m e t h y l c h r o m o n e are p r o d u c e d f o l l o w i n g w o u n d i n g or treatment w i t h ethylene (Coxon et al., 1973). From their structures one can surmise that these isocoumarins and chromones are l ike ly not effective signal compounds, but they may effect growth of A. tumefaciens. Interest i n these or related stress compounds stems f rom their toxic properties. 4 4 O H O R=H, 5,7-dihydroxy-2-methylchromone R=OMe, 5-hydroxy-7-methoxy-2methylchromone R=H, 6-hydroxymellein, R=Me, 6-methoxymellein Scopoletin Figure 1.12 A coumarin, isocoumarins and chromones produced by carrot tuber tissue in response to wounding. Typical ly , it seems that compounds produced i n the greatest abundance, or those possessing striking biological activity are the first to be identified. Acetophenones and hydroxybenzoic acids may be synthesized f rom phenylpropanoid precursors as indicated in Figure 1.13. N e i s h (1964) briefly discussed the l i k e l y biosynthetic routes to the acetophenones p i c e i n , pungenin, and androsin. A simple decarboxylation of a phenyl substituted 8-keto acid appears to be involved. C-6-Ci aldehydes may be produced by reduction of the corresponding benzoic acids, or they may be synthesized before the acids, as has been shown i n certain fungi (Wat and Towers, 1979). V e r y recently, Funk and Brodelius (1990) pointed out that the biosynthetic pathway to vani l l in and other C6-C1 compounds i n plants has not yet been established. V a n i l l i n may be formed from f e r u l o y l - C o A through a 6-oxidation-like process. A. tumefaciens is sensitive to most of the structures i n the p a t h w a y f o l l o w i n g the p r o d u c t i o n of f e ru l i c a c i d f r o m 3,4-dihydroxycinnamate. The application of the metabolic inhibitor cycloheximide has shed some l ight on the biosynthesis of A S i n wounded tobacco (Stachel et ah, 1985b). It was demonstrated that metabolically active plant cells (apparently directly adjacent to the w o u n d site) were required for the production of large amounts of A S . O n the basis of this result it seems certain that a detectable level of de novo synthesis occurs. In Chapter 3, G C - M S analyses reveal that m e d i u m extracts f rom cycloheximide-treated Nicotiana leaf sections were depressed i n , but not devoid of acetophenones. 4 6 Figure 1.13 Possible biosynthetic routes f rom phenylalanine to methyl syringate (A) , H O - A S (B), and A S (C). Enzymes: (1)=PAL,(2) cinnamate-4-hydroxylase, (3),(5) and (14)=hydroxylases, (4) and (6)=0-methyl transferases, (7)=CoA ligase, (13)=decarboxylase. 1.3.6 Vacuolar phenolics A wide variety of plant phenolics are known and many of these are present as water soluble glycosides in vacuoles. It has long been considered that glycosylation serves a dual role i n deactivating the phenolic nucleus and p r o v i d i n g water solubility (Harborne, 1979). Glycosylation of vir- i n d u c i n g phenolics renders the phenolic inactive by blocking the phenolic h y d r o x y l from interacting wi th the bacterial receptor or sensor. For example, for wide host range Agrobacterium tumefaciens, coniferin is inactive whereas the aglycone, coniferyl alcohol, is a strong inducer of vir gene expression (Spencer and Towers, 1988). Cases are known i n which phenolic glucosides released from wounded plant tissue are acted upon by plant B-glucosidases to y i e l d bioactive aglycones. A wel l k n o w n example of this is the occurrence of phlor idz in and its fungitoxic aglycone phloretin (Overeem, 1976). Interestingly, the examples i n the literature include compounds which readily oxidize to toxic quinones, e.g. juglone f rom Juglans nigra L . , a classic example of an allelochemical stored as its inactive glucoside (Rhodes, 1985). The process of hydrolysis might apply equally wel l to both antimicrobial phenolics and to phenolics w h i c h act as signal compounds. Thus the signal compounds acetosyringone and methyl syringic acid may be present in the plant as the corresponding B-D-glucosides. In fact, n B u O H extracts treated wi th T F A or B-glucosidase d i d yield CHCl3-soluble, azr-inducing compound (s) (see Chapter 3). It has been reported that the production of acetosyringone from tobacco leaf discs can be greatly decreased by treatment with cycloheximide (Stachel et al., 1985b). For cycloheximide to prevent product ion of active signal compounds from the corresponding glucosides, it w o u l d have to block the 4 8 synthesis of glucosidases fol lowing wounding. However , glucosidases are l ikely to be present before wounding, so some level of hydrolysis might be expected even i n the presence of cycloheximide. p-Hydroxybenzoic , salicylic, and vanil l ic acids are present i n bound form, presumably as glycosides, i n potato culture cells (Robertson et al, 1969). M o r e recently, K l i c k and Herrmann (1988) examined the distr ibut ion of hydroxybenzoic acid glucosides and hydroxybenzoylglucoses (glucose esters) i n a number of plant families. The glucosides are more common than the esters. Salicylic, 4-hydroxybenzoic, vanillic, protocatechuic, and syringic acids are widespread, although in lower concentration than hydroxycinnamic acid derivatives. Picien and androsin (Figure 1.14) are produced by spruce needles, and upon release are rapidly converted to the aglycones, p-hydroxyacetophenone and acetovanillone (Oswald and Benz, 1989). Acetovanil lone is another virulence induc ing phenolic compound (Stachel et a l , 1985; Spencer and Towers, 1988), and so i n inoculation of this species this compound must have an effect on the level of vir gene expression. In Chapter 3, the identification of the glucosides of acetosyringone, acetovanillone (ie. androsin), and their cc-hydroxy derivatives i n unwounded tobacco leaves is described. O-glucose OH O-glucose Picein Pungenin A n d r o s i n F igure 1.14 Structures of acetophenone glucosides p i c e i n , pungenin, and androsin. A n d r o s i n , or H O - A V - g l u c o s i d e , was identified i n intact tobacco leaves (see Chapter 3). 5 0 1.3.7 Repressors of vir expression A recent report demonstrated that vir gene expression is repressed by nitrogenous compounds which are phenolic i n character (Sahi et ah, 1990). The compound D I M B O A , 2,4-dihydroxy-7-methoxy-2H-l,4,-benzoxazin-3(4H)-one (Figure 1.15), from monocots such as maize and wheat, has long been k n o w n for its antifungal, bacteriostatic, and insect antifeedant properties. Sahi et ah (1990) isolated D I M B O A from maize homogenates on the basis of its suppresive effect on air-induction. Als o , they demonstrated that D I M B O A can prevent tumor formation on leaves of Kalenchoe diagramontiana. D I M B O A is one of a number of hydroxamic acids present i n monocots as its glucoside (Wahlroos and Virtanen, 1959). The glucosides are readily hydrolyzed when the structural integrity of the tissue is destroyed (Bailey and Larson, 1989). The aglycone inhibits vir gene induction by acetosyringone, and is l ikely a significant factor i n the resistance of such monocots to infection by A. tumefaciens. A n H P L C examination of hydroxamates f rom maize is described i n Chapter 3. 5 1 O H R ^ R ^ H I DIBOA -> II B O A R 1 = O C H 3 R 2 =H III D I M B O A -> IV M B O A R 1 = R 2 = O C H 3 V D I M 2 B O A -> VI M 2 B O A Figure 1.15 C h e m i c a l structures of D I M B O A and related hydroxamic acids. DIBOA=2,4-dihydroxy-2H-l ,4 / -benzoxazin-3 ( 4 H ) - o n e , D I M B O A = 2 , 4 - d i h y d r o x y - 7 - m e t y h o x y - 2 H - l , 4 , -benzoxazin-3(4H)-one, DIM2BOA=2,4-dihydroxy-6 / 7-dimethoxy-2H-1,4,-benzoxazin-3(4H)-one, BOA=2-(3H)-benzoxazolinone, M B O A = 6 - m e t h o x y - 2 - ( 3 H ) - b e n z o x a z o l i n o n e , M 2 B O A = 6 , 7 -dimethoxy-2-(3H)-benzoxazolinone. 1.4 Concluding remarks The in planta roles of w o u n d - i n d u c e d plant phenolic compounds , w h i c h A. tumefaciens detects as signal molecules liberated by host plants, remain unknown. Clearly, they do not function i n the same way as do the dihydroxy-subst i tuted r ing compounds. Such compounds are extremely sensitive to oxidation and the products are often antibiotic. It must be assumed that A. tumefaciens has evolved the ability to detect compounds which are less toxic and which very many plants produce. It so happens that the k n o w n signal compounds (AS and H O - A S ) had never previously been discovered, probably because we have had to await the means by w h i c h to observe the biological activity of compounds which are produced i n such minute amounts. A t least i n certain cases, the signal compounds are l ike ly present i n host plants as glucosides (see Chapter 3-hydrolysis of Nicotiana n - B u O H fractions). In these instances, w o u n d i n g abolishes the compartmentalized state of the plant cells and brings together plant enzymes (glucosidases) and inactive conjugates (eg. the glucosides) to yield ufr-activating compounds. Chapter 2: Identification of an Agrobacterium signal compound from grapevine cultivars. 2.1 Foreword: A major portion of this chapter has been published: Spencer, P . A . , Tanaka, A . , and Towers, G . H . N . (1990) A n Agrobacterium signal compound from grapevine cultivars. Phytochemistry 29: 3785-3788. The principle author and researcher was Paul A . Spencer, w i t h some early bioassays and contribution of grapevine extracts f rom D r . A k i r a Tanaka (Plantec Research Institute, Japan, supported by a N a t i o n a l Science Foundat ion Grant (DMB-870-4292) to Dr . Eugene Nester, U n i v e r s i t y of Washington). Otherwise, the research was conducted i n the lab of Dr. G . H . N . Towers. Dr . E d Neeland synthesized H O - A S , and Felipe Balza operated the G C - E I M S . Grapevine samples were provided by Dr. George Eaton (Plant Science, U.B.C.) and use was made of the U . B . C Chemistry Department Mass Spectrometry and N M R Facilities. 2.2 Introduction A range of plant phenolic compounds have been demonstrated to be i n v o l v e d i n induct ion of Agrobacterium tumefaciens v i rulence genes. Initially, acetosyringone, A S , and oc-hydroxyacetosyringone, H O - A S , were identif ied as wide host range (WHR) signal compounds from transformed tobacco root cultures (Stachel et al, 1985). Pr ior to this w o r k , these acetophenones were not k n o w n as natural products. It was subsequently 5 4 demonstrated (Bolton et al, 1986; Spencer and Towers, 1988) that the more c o m m o n p h e n o l i c s , such as certain p h e n y l p r o p a n o i d s ( i n c l u d i n g monolignols) and benzoic acid derivatives, also showed significant vir-inducing activity. In this initial study, a W H R strain of A. tumefaciens was studied; this chapter examines air-induction in a l imited host range (LHR) A. tumefaciens s t ra in . Certain L H R strains of Agrobacterium (eg. A856, Thomashow et al, 1981) are capable of inducing tumors on various grapevine cultivars, to the exclusion of typical hosts (eg. Nicotiana tabacum). W i d e host range (WHR) strains (eg. A348, Thomashow et al, 1981) can induce tumors on a wide variety of dicotyledons. However , W H R strains can cause a hypersensitive response on stems of certain grapevines and typically are unsuccessful at tumor induction on these cultivars (Yanofsky ef al, 1985). One cultivar, Vitis cv. Seyval, is a permissive host for both strains. Interestingly, V. labruscana cv. Steuben is a host only for L H R A. tumefaciens. The inabil i ty of W H R strains to form tumors on grapevines may be due to the hypersensitive response caused by W H R Agrobacterium. Factors both wi th in the T - D N A as wel l as the vir region have been shown to play a role i n host range. Hoekema et al (1984) analysed factors that confer l imi ted host range on strain LBA649, which contains the octopine plasmid pTiAg57. A wide host range T-region gene was found to compliment the host range "defect" in pTiAg57, but a wide host range vir region failed to extend the host range of LBA649. However, this is not the case w i t h all L H R strains studied. Determinants wi th in both the T - D N A and vir region were found to contribute to the host specificity of L H R Agrobacterium possessing p T i A g l 6 2 (Yanofsky et al, 1985). Experimental introduction of W H R vir A and virC functions were required to confer upon the recipient L H R strain an expanded host range. This was an important observation because the V i r A gene product is considered to be the environmental sensor of plant derived signal compounds, and so artificial expansion of host range by introduction of a W H R vir A gene suggested that host range may be determined on the basis of signal molecule specificity. Thus, a factor that may influence host range specificity is the abil i ty to detect the appropriate chemical signal by the bacterial receptor protein. For example, the l imited host range p i r - inducer m a y be some c o m p o u n d other than the p r e v i o u s l y m e n t i o n e d acetophenones. M a et al. (1987) examined thirteen strains of A. tumefaciens isolated from grapevine tumors i n C h i n a . The D N A i n these strains exhibited "little or no homolgy to a W H R vir A locus, but d i d show strong homology to a L H R vir A locus"; a W H R vir A probe failed to hybridize, and a L H R vir A probe strongly hybr id ized w i t h vir A loci f rom the grapevine strains. This supports the concept of a specific vir A locus amongst Agrobacterium strains associated w i t h grapevines. In addit ion, the putative sensors of the phenolic signal compound(s), the L H R and W H R vir A gene products, have been sequenced and compared (Leroux et al., 1987). The gene products were determined to have diverged to the greatest degree i n their assumed periplasmic domain , thus lending support to the concept that differing signal compounds may be required for vir induction i n L H R and W H R strains. For the purpose of understanding the mechanism(s) by w h i c h host ranges are established, investigations of the phytochemicals involved i n vir-induct ion are necessary. In this Chapter is described the bioassay-directed isolation of a single a i r - inducing substance present i n various grapevine-derived w o u n d exudates and its identification as the methyl ester of syringic acid. Neither H O - A S nor AS was found in these extracts. 5 6 2.3 Methods 2.3.1 Plant material Samples of leaves, stems and w o u n d eudates f rom Vitis cultivars Seyval , Foch, Pinot N o i r , DeChaunac, Vidal256, M u l l e r Thurgau , L e o n M a l l o t , W h i t e D i a m o n d , and H i m r o d were obtained f rom the U . B . C . Botanical Gardens and South Campus vineyard.-. 2.3.2 Extraction and Isolation Fresh leaves and stems (0.5-1.0 k g per batch) were cut into small (ca. l cm) pieces and incubated i n 2.0 L filter-sterilized, p H 5.7 M S medium for 24-48 hrs. The filtered medium was partitioned with at least 3 volumes each of CHCI3, CH2CI2, E t O A c , and or n - B u O H . The organic solvents were removed w i t h a rotory evaporator and the extracts resuspended i n small volumes of M e O H . The conditioned tissues were extracted thoroughly w i t h 70% M e O H , the M e O H removed with a rotory evaporator and partitioned against solvents as described above. The crude extracts were fractionated on Sephadex LH-20 columns us ing acetone, M e O H , or M e O H / C H C l 3 . Act ive fractions were pooled, resuspended i n H P L C grade M e O H , and separated further by semi-preparative H P L C (LiChrosorb RP-18, 3 m L / m i n . , 5% aq. H O A c / M e O H ; 30-100% M e O H gradient/30min.). H P L C fractions were extracted w i t h CHCI3, rebioassayed, and the recovered materials i n active H P L C fractions was further separated by T L C on silica [1. E t O A c , 2. C H C l 3 : M e O H , (9:1), 3. C l C H 2 C H 2 C l : C H 3 C O C H 3 , (95:5), 4. toluene:EtOAc:HOFm, (5:4:1)] or polyamide [EtOFm: cyclohexane: BuOAc: H O F m , (50:25:23:2)]. Where possible fractions were stored at or below 0 °C i n H P L C grade M e O H or E t O A c , and reduced i n volume under a stream of N2. 2.3.4 zn'r-induction assays The plate assay system was developed from the system reported by Bolton et al. (1986). In plate assays, a few uL of each test fraction or compound i n M e O H , were spotted onto MeOH-washed 5 m m absorbent fiber discs and the solvent allowed to evaporate. Test discs were placed on separate sectors of an overnight l a w n culture of either A 8 5 6 / p S M 2 4 3 c d (see below) or A348/pSM243cd (see below) grown on p H 5.5 A B m e d i u m (Chil ton et al, 1974) which contained 100 f i g / m L carbenicillin and ca. 0.1% (600 ug/plate) X -gal (5-bromo-4-chloro-3-indolyl-f3-D-galactopyranoside). This chromogenic substrate for the enzyme (3-galactosidase yields a blue colored product. The plates were incubated at 28 °C unti l blue zones of bacterial growth developed i n the areas surronding discs w i t h active compounds (24 hrs.). Quantitative B-galactosidase assays fol lowing induction (9 hrs . / 28 °C) i n p H 5.5 A B medium (100 | ig/mL carbenicillin) containing 0.1% D M S O , were conducted as described by Mil ler (1972). 2.3.5 Bacterial strains, media and plasmids The L H R (pTiAgl62) and W H R (pTiA6) plasmids and the strains conta in ing them (A856 and A348) have been descr ibed p r e v i o u s l y (Thomashow et al, 1981). A856/pSM243cd carries i n addition to p T i A g l 6 2 , a virB::lacZ gene fusion, pSM243cd (Stachel and Zambryski , 1986; Winans et al, 1986) i n the absence of W H R vir A and is thus an indicator of L H R virA-dependent pzr-induction. Conversely, strain A348/pSM243cd carries a vir B:\lac Z gene fusion (pSM243cd) in the presence of the W H R plasmid p T i A 6 and is thus an indicator of W H R t;z>A-dependent pzV-induction. 5 8 2.4 Results and Discussion The plate/disc bioassay system described i n section 2.3.3 was developed i n order to f o l l o w a i r - i n d u c i n g ac t iv i ty f o l l o w i n g each stage of chromatographic fractionation. This assay system was sensitive to 0.5 u,g of A S . Extracts of unwounded grapevine stem or leaf tissue d i d not exhibit any a i r - i n d u c i n g activity, confirming the requirement for w o u n d i n g prior to tumor induct ion by Agrobacterium. However , the stem and leaf sections produced and exuded into the culture medium detectable amounts of air-i n d u c i n g substance, w h e n incubated for 24-48 hrs. i n M S m e d i u m . Interestingly, both A348/pSM243cd and A856/pSM243cd (see methods) responded positively to grapevine extracts i n plate assays. The active, w o u n d -induced c o m p o u n d was found to partition from the condit ioned culture m e d i u m and methanolic tissue extracts into the CH2CI2 or C H C I 3 phase. Prel iminary H P L C and TLC/bioassay experiments w i t h extracts f rom each of the grapevine cultivars suggested that a single compound, common to al l the grapevine cultivars, was l ikely responsible for the observed activity. Furthermore, the same compound was present i n extracts f rom both the w o u n d e d stem tissue and the conditioned m e d i u m , al though i n m u c h greater puri ty i n the latter. Extracts from a number of cultivars were pooled i n order to provide sufficient material for both bioassays and structure e lucidat ion. N o attempt was made to quantify the amounts of active compound produced by any of the grapevine cultivars. The active compound was usually wel l resolved by T L C on silica gel, although it was clear that only very small amounts were being eluted from the isolated bands. Fol lowing gel filtration on Sephadex LH-20, reversed phase H P L C , and multistep silica and polyamide T L C sufficient purif ied material remained to permit identification by mass spectrometry (see Table 2.1). 1 H - N M R spectroscopy of the partially puri f ied grapevine derived compound gave methoxy signals (singlets near 4 ppm) and a phenyl proton signal (singlet near 7.3 ppm). L o w resolution electron impact mass spectroscopy (EIMS) indicated the presence of a molecular ion ( M + ) at m/z 212. H i g h resolut ion mass spectroscopy (HR-EIMS) confirmed a molecular ion at m/z 212 (actually 212.0686), and indicated the molecular formula to be C10H12O5. Two strong vir -inducers wi th this composition are oc-hydroxyacetosyringone (Stachel et ah, 1985) and syringic acid methyl ester. The ester was inc luded i n the analysis of the structure-activity relationships of air- induction (Spencer and Towers, 1988). Other fragments included m/z 181 (C9H9O4), m/z 153 (C.8H9O3), m/z 149 (QH5O3). G C - M S of the si lylated derivative of the grapevine-der ived vir -inducer, and comparison wi th that of authentic a - h y d r o x y a c e t o s y r i n g o n e and methyl syringate, unequivocally identif ied the inducer as the latter. M e t h y l syringate becomes monosilylated ( M + 284), whereas H O - A S gains two T M S groups ( M + 356). Since the same compound was obtained f rom a number of Vitis cultivars, including Seyval, and since no other highly active fractions were isolated, I concluded that the most significant natural ly occurring l imited host range signal compound from grapevines is syringic acid methyl ester. To m y knowledge, this was the first report of the natural occurrence of this compound. To investigate further the effects of syringic acid methyl ester on air-induction i n W H R and L H R Agrobacterium, I performed quantitative assays of B-galactosidase activity in bacteria possessing virwlac gene fusions. The degree of vir gene induct ion w i t h increasing concentration of authentic syringic acid methyl ester and A S in W H R and L H R strains is shown in 6 0 Table 2.1 Spectral and chromatographic data used i n the identification of methylsyringate in grapevine w o u n d exudates. Active grapevine compound : H i g h resolution electron impact mass spectrometry (HR-EIMS) m/z : 212.0686 [ M ] + ( C i o H 1 2 0 5 ) , 181.0501 [ M - O C H 3 ] + (C9H9O4), 153.0552 [ M - C O O C H 3 ] + (C8H9O3); calculated for QoH 1 2 0 5 , 212 .0685 ; C9H9O4; 181.0501, C8H9O3; 153.0552. Gas chromatography-electron impact mass spectrometry (GC-EIMS) of T M S derivative m/z : 284 [ M ] + (30), 269 [ M - M e ] + (44), 255 (14), 254 [M-30] + (100), 223 (22), 73 (31). Thin layer chromatography (TLC): Si gel T L C (1) Rf= 0.52, (2) Rf= 0.63, (3) Rf= 0.28, (4) Rf= 0.46; polyamide T L C Rf= 0.78. H P L C R t = 17.0 min . Authentic cx-hydroxyacetosyringone : Proton nuclear magnetic resonance O H N M R ) (CDCI3): 7.20 (s, 2H) , 4.85 (s, 2H), 3.97 (s, 6H). 1 3 C N M R (CDCI3): 196.7 (s, C=0), 147.1 (s), 140.8 (s), 124.8 (s), 105.0 (d), 65.0 (t, CH2), 56.5 (q, OCH3). G C - M S of T M S derivative m/z (rel. int.): 356 [M]+ (27), 341 [ M - M e ] + (44), 254 (45), 253 [M-CH 20TMS]+ (100), 223 (33), 103 [ C H 2 O T M S ] + (27), 73 (88). Authentic syringic acid methyl ester. J H N M R (CDCI3): 7.33 (s, 2H), 5.94 (s, I H ) , 3.95 (s, 6H), 3.91 (s, 3H). EI-MS m/z (rel. int.): 212 (100), 197 (53.5L181 (89), 169 (19.5), 165 (20.6), 153 (45), 141 (72), 123 (40), 108 (32.3), 95 (30), 79 (28 ), 43 (50). G C - M S of T M S derivative m/z (rel. int.): 284 [M]+ (37), 269 [M-Me]+ (59.5), 255 (23.5), 254 [M-30] + (100), 223 (32), 73 (20). Thin layer chromatography (TLC): Si gel T L C (1) Rf= 0.52, (2) Rf= 0.63, (3) Rf= 0.28, (4) Rf= 0.46. H P L C Rt= 17.0 min . 6 1 Figure 2.1. A s was reported for the W H R strain A348/pSM358 (Spencer and Towers, 1988), the methyl ester is a more potent uz'r-inducer than is A S . L H R vir - induct ion does not reach its peak unti l the concentration rises to about 500 U.M A S , or 200-500 u.M methyl syringate. A t concentrations that induce half-maximal W H R vir expression (about 20 u M methyl syringate), the L H R strain was not detectably activated. Also , the L H R strain had only reached half -maximal induction at concentrations that ful ly induced W H R vir gene expression. The relevance of the results presented here to the phenomenon of host range specificity is obscure. Perhaps the L H R strains are s imply l imited to hosts that produce higher levels of signal compounds. Expression of vir genes of both W H R and L H R strains is inducible by both A S and methyl syringate, although the two strains appear to differ i n their respective vir-i n d u c i n g concentration optima. Previously, A S was identi f ied f rom N. tabacum, a host for W H R A. tumefaciens. Methyl syringate is a very strong z;z'r-inducer of both W H R and L H R strains, and yet grapevine cultivars are more susceptible to tumor formation by L H R strains. The concentrations of t u r - i n d u c i n g compounds produced by different host species must be determined. Addi t iona l ly , an area yet to be explored regards the possible presence of grapevine-derived uzr-inhibitors specific to W H R strains. It should be emphasized that only syringic acid methyl ester was found i n grapevine extracts; neither A S nor H O - A S , both acetophenones, was present. There is l ikely some significance to the elaboration of these different classes of uzr-inducing phenolic compounds by the respective host groups. C o i n c i d e n t a l l y , H O - A S and syr ingic ac id methyl ester are isomeric compounds, possessing the same molecular weight and formula, but subtly different structures. Production of A S can be elicited by treatment of N. WHR & LHR A. tumefaciens vir-induction 600 Concentration (u.M) Figure 2.1. m'rB-Induccion i n strains A348 ( W H R ) and A856 ( L H R ) . Each of these strains possesses, i n addit ion to its W H R or L H R -determining T i plasmid, the virBv.lacZ plasmid pSM243cd. Fo l lowing induct ion w i t h either A S or syringic acid methyl ester (Me-syr.) 6-galactosidase activity was assayed according to Mil ler (1972). 6 3 tabacum cell suspension cultures w i t h cellulase (Threlfall and Whitehead, 1988), although the significance of this compound, other than its role as a signal compound, is not known. The biosynthetic origins and roles for these w o u n d response compounds must be investigated. 6 4 Chapter 3. Naturally Occurring W H R Signal Molecules 3.1 Foreword A major portion of this chapter has been accepted for publication: Spencer, P . A . , and Towers , G . H . N . (1991) Restricted occurrence of acetophenone signal compounds. Phytochemistry (in press). The research was conceived of and conducted by Paul A . Spencer i n the laboratory of Dr. G . H . N . Towers. This Chapter describes the application of combined G C - M S to the analysis of the phenol ic components i n part ia l ly p u r i f i e d W H R A. tumefaciens vir gene activating plant w o u n d exudates. G C retention times and mass spectra were used to confirm the identity of the components i n the active mixtures (refer to Chapter 5). Results confirmed that the production u p o n w ounding of elevated levels of oir-activating acetophenones requires metabolically active tobacco plant cells. A d d i t i o n a l l y , this phenomenon appears restricted i n distribution among plants to members of the Solanaceae. In contrast, species from other plant families were found to produce vir-inducing benzaldehydes, and benzoic and cinnamic acid derivatives but none of the acetophenones. 3.2 Introduction Chapter 2 describes the multi-step isolation and the identification of methyl syringate from grapevine cultivars (Spencer et al., 1990). The active component was found to have a molecular weight of 212, and the chemical composit ion C10H12O5. O n this information alone, it seemed l ikely that this 6 5 was the k n o w n inducing compound a-hydroxyacetosyringone. H o w e v e r , GC-mass spectrometry of derivatized grapevine samples and of the authentic a c e t o p h e n o n e e s t a b l i s h e d that the u n k n o w n w a s n o t a -hydroxyacetosyringone, but an isomeric compound, syringic acid methyl ester. Coincidentally, this ester was an important compound included i n the analysis of structure-activity relationship of tnr-induction i n wide host range ( W H R ) Agrobacterium tumefaciens (Spencer and Towers, 1988). Preparative reverse phase (RP-18) H P L C and normal phase silica gel T L C were used extensively to isolate methyl syringate. W o r k on the L H R signal compound resulted i n improved techniques for analysis of u/ 'r- inducing compounds f rom other host and non-host species. The plate bioassay (described in Spencer et al, 1990) was subsequently used to screen for activity the numerous chromatographic fractions derived from other host plants. A l s o , whereas chromatography on silica gel may be convenient for T L C analysis of various extracts and fractions, it appears to irreversibly b ind some phenolic compounds. Therefore, this was abandoned as a means to isolate pzr-inducing fractions. Instead, as w i l l be described i n this chapter, short columns of polyamide were used to provide fractions greatly enriched w i t h mixtures of tn'r-inducing compounds and wi thout significant loss of material. Fractions prepared i n this way were ready for derivatization and GC-mass spectrometry. Pr ior to its discovery as a signal compound by Stachel et al. (1985), acetosyringone was not known as a natural product. It was conceivable that only Ri-transformed N. tabacum synthesized this novel compound - a quirk of secondary metabolism in an unusual cell culture environment. However , Threlfall and Whitehead (1988) have shown that acetosyringone is produced by N. tabacum suspension cultures treated w i t h cellulase. This result 6 6 suggested either that the acetophenone is a natural cell w a l l constituent or that its p r o d u c t i o n is otherwise el icited by the action of cellulase. Coincidental ly, pretreatment of Panax ginseng callus tissue w i t h cellulase has been used to acheive transformation w i t h Agrobacterium rhizogenes (Yoshikawa and Furuya, 1987). In this case, however, the cellulase was part of a protoplasting treatment, and the protoplasts were wel l washed pr ior to inoculation. Such washing w o u l d l ikely remove any a i r - inducing phenolic compounds released from cell walls by the cellulase. G C - M S analysis of the "conditioned" culture m e d i u m from stem and leaf sections of Nicotiana species confirmed that acetosyringone and its a -hydroxy derivative are produced by nontransformed tissues. Moreover, i n all tobacco extracts the corresponding pair of guaiacyl-substituted acetophenones (acetovani l lone and oc-hydroxyacetovani l lone) were also detected. A d d i t i o n a l l y , the far more commonly occurring phenylpropanoid signal compounds were absent. In this chapter, wound- induced product ion of acetosyringone and other signal compounds from a variety of plant species was investigated. The biosynthesis of signal compounds was also investigated by G C -mass spectrometry. T w o experiments designed to establish the biosynthetic o r i g i n of the acetophenones were conducted. In one experiment, the glycoside fractions from non-wounded plants were prepared. The glycoside fraction exhibited no air- inducing activity. These fractions were hydrolysed either w i t h B-glucosidase or w i t h I M T F A (trifluoroacetic acid) and the hydrolysates tested for activity. In another experiment, the eucaryotic metabolic inhibitor cycloheximide was tested for its ability to inhibit w o u n d -induced product ion of acetophenones from phenylpropanoid precursors. Under these conditions, the presence of the phenylpropanoids instead of the 6 7 acetophenones w o u l d lead one to suspect that the required enzymes are produced fol lowing wounding. 3.3 Methods 3.3.1. air-Induction assays Plate assays were conducted much as described i n Chapter 2, except that here, care was taken to determine concentrations of the extracts. In these assays, 10-40 U.L of 1 m g / m L solutions of each test fraction or compound i n M e O H , were transferred to M e O H - w a s h e d 5 m m absorbent discs and the solvent al lowed to evaporate. Test discs were placed on separated sectors of an overnight lawn culture of A348/pSM243cd grown on p H 5.5 A B medium w h i c h contained 100 u g / m L carbenici l l in ( A B / c b 1 0 0 ) and ca. 0.1% (600 |ig/plate) X-ga l (5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside). The plates were incubated at 28 °C unt i l blue zones developed i n the areas surrounding discs wi th active compounds (24 hrs.). The plates were also examined after an additional 24 hrs. for late developing fractions. Quantitative B-galactosidase assays fo l lowing induct ion (8-20 h r s . / 28°C) i n M E S buffered p H 5.5 A B medium containing 0.1% D M S O and 100 u.g/mL carbenicill in, were conducted as described by M i l l e r (1972). W h e n quantitatively assaying the effect of pure compounds, D M S O was added to dissolve the crystals and filter sterilized A B / c b 1 0 0 m e d i u m added to make stocks solutions (eg. 200 uM) containing 0.1% D M S O . Control experiments were conducted to check the effects of D M S O (and ethylene glycol monoethyl ether). This method works equally wel l for both A. tumefaciens and A. rhizogenes A4/pSM358cd (see Chapter 4). 6 8 3.3.2. Plant materials A t the outset of this project, the need for a "signal compound survey" was proposed. In other words, a variety of host and nonhost species should be examined for any of a number of biologically active phenolic compounds. A t that time there was no established means by which such a survey could be pursued. N o rapid bioassay or standardized chromatographic methods were k n o w n . W o r k on the isolation of the grapevine-derived signal compound (Chapter 2) resulted i n standardized methods (notably polyamide V L C and G C - M S ) which have been used here i n accumulating data on a range of plant species. So far, the conditioned media from plant parts and cell cultures representing 41 species have been examined for CHCl3-soluble afr-inducers (Table 3.1). Seedlings of Nicotiana species were k i n d l y prov ided by the Agricul ture Canada Research Station at U . B . C . These were brought to the Botany Department greenhouse and grown there unti l required. Most other species were available from the U.B .C . Botanical Gardens, endowment lands or South Campus area. 3.3.3. H P L C of maize hydroxamates The E t O A c part from the aqueous medium conditioned by wounded corn stem sections was filtered, reduced to dryness under N 2 and resuspended i n H P L C grade M e O H . A semipreparative LiChrosorb RP-18 (Merck) H P L C column was equilibrated with 15% M e O H / 85% l O m M H3PO4 at a f low rate of 3 m L / m i n . and 100 uL aliquots were fractionated using gradient elution starting at 15 % for 4 min . , increasing to 60% in 25 m i n . , and then to 100% M e O H i n 10 min . Compounds were detected by absorbance at 265 nm. 6 9 Table 3.1 Species examined for CHCb-soluble air-inducers Plant species Fami ly Source of inducers Ambrosia chamissonis (Asteraceae) root culture m e d i u m Arabidopsis thaliana (Brassicaceae) stems Asparagus officinale (Liliaceae) stems Atropa belladonna (Solanaceae) leaves Beta vulgaris (Cheanapodiaceae) tubers Brassica sp. (Brassicaceae) stems Capsella bursa-pastorus (Brassicaceae) stems Chaenactis douglasii (Asteraceae) culture m e d i u m Datura stramonium (Solanaceae) leaves Daucus carota (Apiaceae) tubers Discorea (?) (Dioscoraceae) tubers Geranium richardsonii (Geraniaceae) stems Hydrangea sp. (Hydrangeaceae) stems Hyoscyamus niger (Solanaceae) leaves Lolium perene (Graminea) crown region Lycopersicon esculentum (Solanaceae) leaves Kalanchoe diagramontiana (Crassulaceae) leaves Mentha sp. (Lamiaceae) stems Nepeta cataria (Lamiaceae) stems Nicotiana tabacum (Solanaceae) stems+leaves Nicotiana glauca (Solanaceae) stems+leaves Nicotiana plumbaginofolia (Solanaceae) stems+leaves Nicotiana rustica (Solanaceae) stems+leaves Nicotiana silvestris (Solanaceae) leaves Nicotiana clevelandii (Solanaceae) leaves Nicotiana glutinosa (Solanaceae) leaves Panax gensing (Araliaceae) culture m e d i u m Picea sitchensis (Pinaceae) stems+needles Polygonum aubertii (Polygonaceae) stems Populus H y b r i d H H (Salicaceae) stems+petioles Pseudotsuga menzeisii (Pinaceae) stems Rosa sp. (Rosaceae) stems Solanum dulcimara (Solanaceae) leaves Solanum tuberosum (Solanaceae) tubers Taxus baccata (Taxaceae) needles Taxus brevifolia (Taxaceae) culture m e d i u m Thuja plicata (Cupressaceae) needles Trifolium repens (Fabaceae) stolons Vitis vinifera (9 cv.'s) (Vitadaceae) stems+leaves Zea mays (Poaceae) crown region Zingiber officinale (Zingiberaceae) rhizomes 7 0 3.3.4. Bioassay sample preparation Stem, stolon, rhizome or tuber sections f rom fresh plant material approximately 0.5-1.0 cm i n length (or about 1.0 c m 2 ) were prepared under semisterile conditions and immediately immersed i n about 400 m L filter sterilized M S medium (15 g / L sucrose) at p H 5.7, without added buffer. In each case the wounded plant material was incubated at about 25 °C for 24 hrs. w i t h continual shaking (100 rpm) i n relative darkness. The condit ioned m e d i u m from each plant species was filtered, and the final p H was recorded. The condit ioned m e d i u m was partitioned w i t h at least three volumes of disti l led C H C I 3 , CH2CI2 or EtOAc. The organic phase was reduced to dryness w i t h a rotory evaporator and resuspended i n H P L C M e O H to give solutions of 20 m g / m L . 20-40 uL of each extract was assayed for air-induction by disc-plate bioassay w i t h Agrobacterium as described. 3.3.5. Polyamide V L C chromatography The i n i t i a l bioassays of crude CHCI3 or C H 2 C 1 2 fractions f r o m conditioned media d i d not always give strongly positive responses and G C -M S revealed complex chemical compositions. Excellent purif ication of vir-inducers has been achieved using a variation of a technique intended for r a p i d isolation of plant alkaloids (Pelletier et al, 1986). V a c u u m - l i q u i d chromatography ( V L C ) uses a gentle vacuum to d r a w solvent mixtures through columns of T L C grade sorbent. Small columns of T L C grade polyamide were prepared i n 2.5 cm funnels with scintered glass filters. These columns were pre-washed w i t h solvents ( M e O H to CHCi3 :Hexane) us ing vacuum to draw the solvents through. A glass filter was applied to the top of each column to prevent disturbance of the polyamide as additional solvents were added. The extracts (usually only 10-20 mg) were applied i n a minimal 7 1 amount of the first solvent and components eluted w i t h the f o l l o w i n g solvent mixtures: CHCl3:hexane CHCi3:hexane C H C l 3 : M e O H M e O H (1:1) (9:1) (9:1) (neat) 2x~20mL 2x~20mL 2x~20mL 2x~20mL The fractions were reduced to dryness and resuspended (to 10 m g / m L ) i n H P L C M e O H for bioassay. A t this stage only 10 uL, ie. only 100 u\g, of each fraction was applied to a filter paper disc for plate assay. This system usually resolved inducing compounds into one or two fractions (usually the first two solvent mixtures). S u b s e q u e n t - G C - M S analysis indicated that, compared w i t h crude extracts, the complexity of these samples was greatly d iminished, i.e. the number of G C peaks was significantly reduced. Further purif ication was unnecessary. 3.3.6. Hydrolysis of glycosides The n-BuOH-soluble fraction of hot aqueous 60 % M e O H leaf extracts of N. glauca or N. silvestris were reduced to dryness and the residue was resuspended i n p H 5.0 acetate buffer. This was incubated for 24hrs. w i t h 6-glucosidase (Sigma™). The hydrolyzed sample was diluted wi th distilled H 2 O and part i t ioned w i t h dist i l led CHCI3. The organic phase was treated by polyamide V L C for aglycones as described above. The active fraction was examined by G C - M S . Similarly, samples of the n - B u O H extracts were hydrolyzed w i t h 1 M T F A (trifluoroacetic acid) and subsequently partitioned for aglycones w i t h 72 distil led CHCI3. Aga in , the organic phase was separated by polyamide V L C for aglycones as described above. 3.3.7 Cycloheximide treatment: To examine the effects of cycloheximide on the production of signal molecules, 20 u M cycloheximide was added to the M S culture m e d i u m prior to the addit ion of N. sylvestris leaf sections. Otherwise, the sections were incubated for 24 hrs. under the same conditions as were non-cycloheximide treated leaf sections. The medium was then filtered, partitioned w i t h CHCI3 and analysed i n the same way as were extracts f rom non-cycloheximide treated tissues. 3.3.8 Cellulase-induced release of inducers from Nicotiana cell walls Solvent extracted cell w a l l material from N. silvestris (100 mg) was suspended i n 50 m L p H 5.0 acetate buffer and incubated at 37 °C overnight w i t h a mixture of cellulase and Drieselase. The supernatant was partitioned w i t h CHCI3, resuspended i n M e O H to 1 m g / m L and tested for activity by plate-bioassay. 73 3.4 Results and Discussion 3.4.1 Signal compound mixtures W o u n d induced plant phenolic compounds exhibi t ing b io logica l activity i n Agrobacterium strain A348/pSM243cd were successfully pur i f ied f rom leaf, petiole, stem, root, rhizome or tuber section-conditioned culture media by solvent partitioning and column chromatography. The polyamide V L C system usually resolved vir gene activating mixtures into one or two fractions (usually the first or second two solvent mixtures). In this way, for some samples, about 95% by mass of inactive material i n the extracts could be removed prior to G C - M S . Because very small amounts of material were obtained, only rough estimates of the quantities of compounds were obtained. Based on plate assay responses equivalent to that of 0.1-10 |ig A S , the range of concentrations of inducing substances produced by the Nicotiana species i n 24 hrs. was approximately 1.0-10 ug A S eq./g.f.w. leaf tissue. G C - M S data indicated the presence of a number of virulence inducing phenolic compounds i n wounded plant conditioned media. G C - M S analysis of partially purif ied extracts is an ideal means by which a number of active compounds i n mixtures can be identified. Individual components were not subjected to mul t ip le chromatographic steps d u r i n g their i solat ion, a procedure w h i c h leads to loss of material at each stage. The f o l l o w i n g example demonstrates the usefulness of G C - M S analysis i n these studies. Fo l lowing gel filtration, extensive silica gel T L C and H P L C , methylsyringate was isolated from wounded grapevine conditioned media (Chapter 2; Spencer and Towers, 1990). U s i n g G C - M S of relatively crude grapevine extracts syr ingaldehyde and syringic acid were detected i n addi t ion to methyl syringate. Apparent ly the aldehyde and the acid were lost d u r i n g the isolation of the methyl ester. The trimethylsilyl (TMS) derivatives of these phenols typically give strong molecular M + ions, and fragmentation patterns often exhibited strong [M-15] + , [M-30] + , and [M-45] + ions. [M-31] + and [M-61]+ ions were noted i n mass spectra of benzoic acid methyl esters. [M-89] + and [ M -119J + ions were observed i n mass spectra of ferulic and sinapic acids. Ions of m/z 45, 59, 73, and 89 were very common. Trends i n the dis tr ibut ion of signal compounds elaborated f r o m representative plant families were observed (Table 3.2). Differing groups of signal compounds were revealed by G C - M S analysis. The common phenolic acids vanil l ic , syringic, ferulic, and sinapic acids were present i n many of the extracts f rom w o u n d e d plant tissue conditioned media. In certain media other k n o w n inducers were detected. These included the methyl esters of vani l l i c and syringic acids (eg. grapevine cultivars). Yet another group yei lded vani l l in , and coniferyl alcohol ( eg. conifers) upon wounding. The differing arrays of virulence inducing compounds produced upon w o u n d i n g m a y be used to arrange v a r i o u s p lant species into chemotaxonomic groups. Certain conifers do produce some active phenolic compounds, but they include vani l l in and i n one case, coniferyl alcohol. Every solanaceous plant examined, including all species of tobacco, produced guaiacyl and syr ingyl substituted acetophenones and their a - h y d r o x y derivatives (section 3.4.2). Grapevine cultivars produced benzoic acids and their m e t h y l esters. M a n y dicots and monocots p r o d u c e d active phenylpropanoids (cinnamic acid derivatives) as w e l l as benzoic acid derivatives. The active compounds present in a very active sample obtained from m e d i u m conditioned w i t h Phaseolus sp. (Scarlet runner) leaf sections could Table 3.2. The signalling phenolics detected by G C - M S of w o u n d exudates f rom selected species of f lowering plants. The signal molecules by number are: 1=AS, 2=HO-AS, 3=AV, 4 = H O - A V , 5=vani l l in , 6=syringaldehyde, 7=vanillic ac id , 8=syringic ac id , 9=ferulic acid, 10=sinapic acid, l l = m e t h y l vanil late, 12=methyl syringate, 13=coniferyl alcohol. + = compound detected, +/- = compounds at detection l imit , - = compound not detected. Plant aenus & family Signal c o m p o u n d number 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 Monocotvledonae Asparagus Liliaceae +/-Dioscorea Dioscoraceae + + + + - - -Lolium . Poaceae + + + + - - -Zea Poaceae + + + + - - -Zingiber (rhizome) Zingiberaceae + + + + - - -Dicotyledonae Arabidopsis Brassicaceae +/- +/-Brassica Brassicaceae + + + + - - -Capsella .Brassicaceae + + + + - - -Daucus (tuber) . Apiaceae + + + + - - -Geranium Geraniaceae + + + + - - -Helianthus - - - - +/- +/- - - +/- - +/- - -Hydrangea Hydrangeaceae +/- +/- +/-+ + + + - - -Phaseolus Fabaceae Populus Salicaceae + + + + - - -Rosa Rosaceae + + + + - - -Vitis Vitidaceae + + + - - - + -Atropa Solanaceae + + + + + - - + - -Datura . Solanaceae + + + + + - - - - - + - -Hyoscyamus ... , Solanaceae + + + + + - - - - - + - -Lycopersicon ... ., Solanaceae + + + + + - - - - - + - -Nicotiana Solanaceae + + + + + - - - - + - -Scopolia Solanaceae + + + + + - - - - - + - -Solanum + + + + + - - - +/- - + - -Solanum (tuber) ...Solanaceae + + + + - - -Coniferae » Picea Pinaceae Pseudotsuga.... Pinaceae - - - - + + Taxus Taxaceae + Thuja , Cupressaceae +/• 7 6 not be identif ied. None of the 13 compounds indicated i n Table 3.2 were detected from bean leaves by G C - M S . The Fabaceae are wel l k n o w n for the variety of flavonoids they produce. Also , 4,4'-dihydroxy-2'-methoxychalcone is k n o w n both as a stress metabolite from Pisum sativum (Car lson and D o l p h i n , 1982) and as the n o d - i n d u c i n g s ignal molecule f r o m alfalfa ( M a x w e l l et al, 1989). These facts lead me to suspect that the Phaseolus sample contained a air-activating chalcone derivative(s). This species should be reexamined, and other members of the Fabaceae also, should be examined i n particular for a ir - inducing chalcones. Identification of legume-derived chalcone s ignal molecules w o u l d demonstrate homologous s i g n a l l i n g systems i n A. tumefaciens and Rhizobium species. Perhaps it w i l l be found that chalcone signal molecules are restricted i n occurrence to members of the Fabaceae, just as acetophenone signal molecules appear to be restricted i n occurrence to members of the Solanaceae (described below). 3.4.2 Solanaceous air-inducers F o l l o w i n g w o u n d i n g , the Nicotiana species produced a pair of acetophenones and their oc-hydroxy derivatives. The two k n o w n inducers acetosyringone (AS), and a-hydroxyacetosyringone ( H O - A S ) , as w e l l as acetovanillone (here called " A V " ) and a newly described c o m p o u n d , a -hydroxyacetovanil lone (a-hydroxy-3-methoxy-4-hydroxyacetophenone, or " H O - A V " ) , were detected. Authentic standards were used to confirm the identities of A S , H O - A S and A V . H O - A V was identified by mass spectral s imilar i ty w i t h authentic H O - A S (Spencer and Towers, 1990). The mass spectrum of the bis-TMSi derivative of H O - A S (mfz 356) exhibits l o w relative abundance M + and [M-15] + ions and a very strong [M-103] + fragment ion (base peak). This l ikely represents loss of [CH2O-TMS] from the molecular ion. 7 7 This pattern was also observed i n the mass spectrum of H O - A V (m/z 326). Both leaf and stem sections produced these compounds. Leaf sections produced a greater amount of tnr-inducers per g.f.w. Nicotiana glauca, a host for both wide and l imited host range Agrobacterium strains (Yanofsky et al, 1985), also produced the acetophenones. W o u n d e d leaves of Lycopersicon esculentum, Solanum dulcamara, S. tuberosum, Datura stramonium, Atropa belladonna, Hyoscyamus niger, and Scopolia japonicum each produced the four acetophenones i n v a r y i n g proport ions. F r o m responses i n plate bioassays the total activity i n extracts could be estimated i n terms of the equivalent activity of pure A S . Thus, lycopersicon esculentum leaves produced at least 5.5 |ig A S eq./g.f .w. N. glutinosa leaves produced about 9.5 |ig A S eq. /g. f .w. G C - M S revealed that sinapic and ferulic acids were not present i n the active mixtures derived from these solanaceous plants. The benzoic acid derivatives, vani l l ic and syringic acids, were also generally absent. Vani l l in and methyl vanillate were often present. Ethyl acetate fractions from media conditioned w i t h stems or leaves of N. glauca d i d not significantly induce virulence. In fact, 2 m m clearing zones were observed surrounding test discs, indicating the presence of a growth inhib i tor . This was not further characterized. H o w e v e r , this result demonstrates that both activating and inhibitory substances may be released u p o n w o u n d i n g . The absence of the common cinnamic and benzoic acid derivatives suggests a general shunt of phenylpropanoid precursors towards C6-C2 compounds. It seems that the enzyme systems required for the synthesis of the acetophenones are switched on to high levels by w o u n d i n g only i n the Solanaceae (see Table 3.2). However, it should be stressed that some level of acetophenone synthesis occurs in the unwounded plant. The presence of the 78 acetophenone-glucosides i n intact tobacco leaves (section 3.4.3) may s imply represent a background level of biosynthesis i n the absence of obvious w o u n d i n g , or may reflect a small but important pool of glucosides ready for hydrolysis upon tissue damage. 3.4.3 Biosynthetic precursors of acetophenones i n the Nicotiana 3.4.3.1 Glycosides as biosynthetic precursors M a n y of the k n o w n plant phenolics are present i n plant cells as water soluble glycosides contained wi th in vacuoles. It has long been considered that glycosylation serves a dual role i n deactivating the phenolic nucleus and p r o v i d i n g water solubility (Harborne, 1979). Glycosylation of a i r - i n d u c i n g phenolics renders the phenolic inactive by blocking the phenolic h y d r o x y l from interacting wi th the bacterial receptor or sensor. For example, coniferin is inactive whereas the aglycone, coniferyl alcohol, actively induces vir gene expression (Spencer and Towers, 1988). It seemed l ikely that signal compounds such as acetosyringone and methyl syringate are present i n the plant as the corresponding glucosides. In fact, n - B u O H extracts from Vitis leaf-conditioned medium, when treated w i t h T F A or (3-glucosidase also yielded CHCi3-soluble, a ir- inducing compound(s) (data not shown). A search for pre-existing acetophenone glycosides was conducted by extraction of non-wounded tobacco leaves w i t h hot 60 % methanol , hydrolysis w i t h dilute T F A or emulsin (see methods), solvent partit ioning for aglycones, polyamide V L C , trimethylsilyl derivit ization and G C - M S . For N. glauca, an estimated 100 ng A S eq. /g. f .w. is present before wounding as glycosides. Fol lowing 24 hr. incubation of leaf sections, the level of a ir - inducing aglycones recoverable from the culture medium increased to about 2.5 pig A S eq. /g . f .w. These data were estimated from plate assay responses, however we believe that it reflects a significant increase (ca. 25-fold) i n the level of inducer compounds over glucoside precursor levels. The results suggest that a certain amount of the active aglycones A S and A V , as wel l as their a-hydroxy derivatives, may be derived from the corresponding glycosides by hydrolys is f o l l o w i n g w o u n d i n g . H o w e v e r , the amounts present i n intact leaves prior to wounding is not sufficient to account for the amount of aglycones recoverable from conditioned media . Thus , n e w acetophenone units must also be generated specifically i n response to w o u n d i n g (see also, section 3.4.3.2). 3.4.3.2 Effects of cycloheximide Stachel et al. (1985) reported that 20 u M cycloheximide i n the plant cell culture m e d i u m prevented production of a i r - induc ing compounds f r o m tobacco leaf discs. In the present study, it was established by polyamide V L C and G C - M S , that 20 u M cycloheximide specifically inhibits production of large amounts of the acetophenones by incubated tobacco leaf sections. The results are i n agreement w i t h those of Stachel et al. (1985b). Apparent ly , de novo biosynthesis of signal molecules does occur. Despite the cycloheximide treatment, a small amount of each of the acetophenone aglycones were detected by G C - M S . For cycloheximide to c o m p l e t e l y prevent p r o d u c t i o n of active s ignal c o m p o u n d s , p lant glucosidases w o u l d have to be newly synthesized f o l l o w i n g w o u n d i n g . However , glucosidase activity of cell walls is wel l established (see Fry , 1988), and this activity l ikely accounts for the production of some of the aglycones. Based on A S equivalents determined from plate assay responses for extracts f rom N. silvestris, an estimated 15-fold higher level of signal compound 80 production was found i n the absence of cycloheximide. These results suggest that cycloheximide prevents formation of new acetophenones, but does not prevent formation of the aglycones from the small pool of glucosides by the action of endogenous B-glucosidase. A l s o , a small amount of de novo acetophenone biosynthesis may result from the action of a l o w level of the required enzymes present before the cycloheximide treatment. The advantage, i f any, to the plants producing these compounds upon w o u n d i n g is not clear. The toxicity of the acetophenones i n comparison wi th the phenolic acids towards other organisms should be investigated. A S is not s t rongly ant i funga l (Threl fa l l and W h i t e h e a d , 1988), and o n l y at concentrations above 200 u M does it inhibi t growth of A. tumefaciens (Spencer and Towers, 1988). It is interesting to note that addition of 50 u M A S and A V inhibit nod gene induction i n Rhizobium leguminosarum by 46% and 85% respectively (Firmin et al, 1986). 3.4.3.3 Cellulase treated cell wal l material The cellulase treatment released a quantity of air-activating substances. It was of interest to establish whether acetophenones such as A S could be released from cell walls by this treatment, ie. whether A S is a component of cell walls i n the Nicotiana. This result w o u l d explain the cellulase-induced product ion of A S i n tobacco suspension cultures reported by Threlfal l and Whitehead (1988). It w o u l d also have been the first report of cell w a l l bound acetophenones i n plants. G C - M S analysis d i d not confirm the release of acetophenones from cell w a l l material, however, both cis and frans-ferulic acid and small amounts of cis and frans-sinapic acid were detected. Perhaps these cinnamic acids are a 8 1 source of precursors i n the product ion of the acetophenones f o l l o w i n g w o u n d i n g . 3.4.4 Inducers and inhibitors from monocots One line of research was to investigate whether there was any clear evidence for the elaboration of differing sets of signal compounds amongst hosts and nonhosts of A. tumefaciens. Since the monocots are generally resistant to infection by Agrobacterium strains, a few representatives were i n c l u d e d i n the survey. Surprisingly, monocot crown region, tuber and rhizome tissues were found to produce a number of the signal compounds elaborated by susceptible hosts. Cultured segments of the rhizome of Zingiber officinale exuded detectable amounts of uir- inducing phenolic acids into the m e d i u m . Transformation of Asparagus officinalis, wi thout a d d i t i o n of exogenous v i ru lence i n d u c i n g c o m p o u n d s , has been demonstrated (Hernalsteens et al, 1984). The active extract from stems of Asparagus officinalis contained methyl vanillate, but none of the other common signal compounds. Zea mays c rown tissue produced the common i ? f r - in duc in g phenol ic acids mixture. Clear ly , other factors, perhaps i n c l u d i n g vir-repressors, wound-induced monosaccharides and p H effects, must play a role i n resistance of monocots to crown gall disease. F r o m w o u n d e d maize condit ioned m e d i u m , z?/r-inducers were obtained i n the CHCI3 soluble fraction, and the az'r-inhibitory hydroxamates D I M B O A and its breakdown products were tentatively identified i n the E t O A c soluble fraction. Under the H P L C conditions described, the hydroxamates were eluted in the fol lowing order: D I M 2 B O A , 22.69 min . ; H M B O A , 23.76 m i n . ; D I M B O A , 24.88 min . ; M B O A , 25.79 m i n . These were tentatively identified by comparison with elution profiles obtained by Dr. Thor Arnason 8 2 (University of Ottawa, personal communication). Thus, upon inoculation of these monocot tissues w i t h A. tumefaciens, the bacteria w o u l d be subject to both air -act ivat ing and a i r - inhib i tory compounds. Another important bacterial growth and virulence factor, p H , is discussed in section 3.4.6. 3.4.5 Conifer extracts Conifers produced other air-activating compounds when w o u n d e d , but, clearing zones were also noted on assay plates. The active compounds produced by wounded Taxus baccata needles and stems included vani l l in and coniferyl alcohol. The inhibitory compounds remain unidentif ied. Perhaps these compounds could be isolated by way of "air-suppression" bioassays. A quinone (2,6-dimethoxyhydroquinone) may have been present i n the active V L C fraction obtained from Taxus baccata (see Chapter 5). The effects of this and other quinones on air- induction should be investigated. Pseudotsuga menzeisii produced very small quantities of vani l l in and syringaldehyde. Picea sitchensis and Thuja plicata produced such small quantities of virulence inducers that G C - M S results were inconclusive. The presence of only l o w levels of air-inducers from conifers is consistent w i t h the fact that conifer species are resistant to crown gall disease. Picien and androsin are produced by spruce needles, and upon damage they are rapidly converted to the aglycones, p-hydroxyacetophenone a n d acetovanillone (Oswald and Benz, 1989). Acetovanillone is another virulence inducing phenolic compound (Stachel et al., 1985; Spencer and Towers, 1988), and so upon w o u n d i n g this species one w o u l d suspect that this compound must have an effect on the level of vir gene expression. C u r i o u s l y , this compound was not found in active V L C fractions from spruce. 83 3.4.6 p H effects In most cases, the p H of the 24 hr. conditioned medium was recorded. Initially, the p H of the M S culture medium (15 g sucrose/L) was adjusted to p H 5.7. In the absence of a buffer system (as w o u l d be the case under natural circumstances), the ability of wounded plant tissues to acidify the culture m e d i u m may greatly effect the degree of air-induction. The f inal p H after incubation of w o u n d e d plant tissues varied by 3 p H units between plant species (Table 3.3). This represents a fairly large range when compared w i t h the opt imum range for air-induction (section 1.2). Stem tissues and leaf tissues from the same plant species differed i n their ability to reduce p H . This was further studied i n the Nicotina species. For each species studied, wounded stem tissue conditioned media were of lower p H than w o u n d e d leaf conditioned media. W o u n d e d N. tabacum stems lowered the p H to a level comparable to that by some monocots and conifer tissues. N. rustica leaves and Solanum tuberosum tuber tissues slightly raised the p H . Data concerning factors such as p H may be important i n understanding crown gall host range phenomena. W o u n d e d tissues that significantly reduce or increase the p H beyond the opt imum for induction of virulence may completely prevent transformation. A number of conifers and monocotyledons acidified the m e d i u m to a level that w o u l d lie outside the optimum p H for a ir - induct ion. H o w e v e r , Rosa species are quite susceptible to crown gall and yet the collection reported here, a w i l d rose species, had a strongly acidifying effect on the culture medium. The lowest p H was recorded for media conditioned wi th wounded A. officinale stem tissue, and yet this is a monocot on w h i c h crown gall tumors can be produced. These last two observations do not support the v iew 8 4 that the abil i ty to reduce the p H of the culture m e d i u m correlates w i t h transformability. 8 5 T A B L E 3.3 Final p H values. Arabidopsis thaliana Asparagus officinale Beta vulgaris Daucus carota Dioscorea Geranium richardsonii Hydrangea sp. Kalanchoe esculentum Lycopersicon esculentum N. glauca N. glauca N. plumbaginafolia N. rustica N. rustica N. silvestris N. tabacum (Samsun) N. tabacum (White burley) N. tabacum (White burley) Picea sitchensis Polygonum aubertii Populus , hybrid H l l Pseudotsuga menzeisii Rosa sp. Solanum dulcimara Solanum tuberosum Solanum tuberosum Taxus bacata Trifolium repens Vitis sp. (cv. DeChaunac) Zea mays Zingiber officinale stems 5.79 stems 3.01 tuber 5.17 tuber 4.74 tuber 4.19 stems 4.48 stems 3.86 leaves 4.64 leaves 4.13 leaves 5.59 stems 4.66 stems 5.25 leaves 6.17 stems 4.49 leaves 5.63 stems 4.10 leaves 5.37 stems 4.10 stems 3.52 stems 4.11 stems 5.60 stems 3.87 stems 3.75 leaves 4.90 leaves 6.02 tuber 6.20 needles 4.47 stolons 4.78 stem sap 5.30 crown 4.54 r h i z o m e 4.53 8 6 Chapter 4: vir expression in A. rhizogenes 4.1 Introduction The analysis of vir gene expression i n Agrobacterium species need not be restricted to wide and l imi ted host range strains of A. tumefaciens. A n o t h e r species of Agrobacterium, A. rhizogenes, is also capable of transforming plant cells, i n this case causing hairy root disease. A s i n crown gall disease, D N A is transferred to susceptible plant cells (Chilton et al, 1982). This very l ikely follows expression of a set of vir genes, although the analysis vir gene expression i n this species has not been reported. The bacteria gain access to susceptible cells through wounds i n plant roots, and presumably w o u n d induced phenolics are responsible for z?ir-induction. The main objective of the experiments described i n this chapter was to ascertain whether the vir genes of A. rhizogenes are i n d u c e d by the phenol ic compounds which induce vir genes of A. tumefaciens. This chapter describes the results of triparental mating experiments and subsequent in>-induction assays w i t h an Agrobacterium rhizogenes strain. The A. rhizogenes strain "A4" has been widely used to generate hairy root cultures of many plant species. The vir region of A rhizogenes is k n o w n to be very similar to that of A. tumefaciens (Hirayama et al, 1988), so it seemed that introduction of a vir-.-.lac gene fusion ("reporter") plasmid into A. rhizogenes w o u l d l ike ly permit analysis of vir gene expression i n this species. The virEr.lacZ fusion plasmid pSM358cd (Winans et al, 1986), which contains the A. tumefaciens virE promoter , was in t roduced into A. rhizogenes A 4 by mating wi th E. coli strains p R K 2013 (helper) and pSM358cd 8 7 (lacZ) (vir) ^ ^ 2 0 1 ^ ^ Agrobacterium rhizogenes A4/pSM358cd (virE::lacZ) Figure 4.1 Introduction of a vir::lac insertion plasmid into A. rhizogenes A 4 by triparental mating. 8 8 (donor) to make the derivative A4/pSM358cd (outlined i n Figure 4.1). The plasmid pSM358cd was chosen because the level of vir activity from its virE promoter is greater than virB reporter gene fusion pSM243cd (Winans et al, 1986) and therefore air-activation may be more accurately quantified. In this chapter, a brief structure-activity analysis of vir gene expression i n the new strain A 4 / p S M 3 5 8 c d , l ike that reported by Spencer and Towers (1988), is described. 4.2 Methods 4.2.1Triparental mating L o g phase cultures of donor, helper and recipient bacterial strains were prepared. The E. coli strains (kindly provided by Dr. Eugene Nester, U .W.) were grown at 37 °C i n LB medium with the appropriate antibiotics. The helper strain (E. coli p R K 2013) was cultured i n the presence of spectinomycin (30 p g / m L ) . The donor strain was cultured i n the presence of carbenicillin and kanamycin (100 j i g / m L ) . The recipient strain "A4" was first screened for resistance to antibiotics that could be used fol lowing mating to select for the recipient (transconjugant). A. rhizogenes A 4 (nal r), grown i n P D B medium at 26° C required a longer culture period than d i d the E. coli strains (freshly prepared P D B from potatoes is recomended). Triparental mat ing was established between the E. coli strains JC 2926/pRK2013 (spec r) and JC 2926/pSM358cd (kan r , carb r) and A. rhizogenes AA (nal r) by mix ing together small swabs of each culture on an LB plate without antibiotics. After 24 hrs. at 28 °C, a sample was streaked out on a P D A selection plate containing kanamycin , carbenicil l in and nal idixic acid. Colonies were selected and g r o w n at 28° C i n P D l i q u i d m e d i u m w i t h the same antibiotics. The 8 9 transconjugant (A4/pSM358cd) was grown i n l iqu id culture and 0.5 m L 50% glycerol stocks stored at -80 °C i n sterile Epindorf tubes. 4.2.2 zn'r-induction assays For znr-induction assays, 10 m L log phase A 4 / p S M 3 5 8 c d cultures i n freshly prepared P D B (potato dextrose broth) w i t h 100 u,g/mL carbenicillin were prepared from glycerol stocks and 100 U.L of culture added to 10 m L of solutions of test compounds i n M E S buffered A B (Chi l ton et al., 1974) m e d i u m at p H 5.5. Quantitative 6-galactosidase assays fo l lowing induction (10 hrs . / 26-2 8 ° C , 150 rpm) i n filter steri l ized p H 5.5 A B m e d i u m (100 u .g /mL carbenicillin) containing 0.1% D M S O , were conducted essentially as described by M i l l e r (1972). When quantitatively assaying the effect of pure compounds, sufficient D M S O was added to dissolve the crystals (roughly 40-60 u,L) and sufficient A B medium added (roughly 40-60 mL) to make 50-400 u M stocks solutions containing 0.1% D M S O . A s was previously reported (Spencer and Towers, 1988), diff iculty was encountered in attempting to resuspend the chalcone derivative to concentrations greater than 50 u M . 4.3 Results and discussion Prel iminary B-galactosidase assays indicated that triparental mating was successful. A 20 hour assay of A 4 / p S M 3 5 8 c d w i t h and wi thout acetosyringone i n p H 5.5 A B medium supplemented w i t h carbenicillin (100 u.g/mL) was conducted at 21 and 28 °C. Some 8000 units of induced activity was recorded. This suggested that A 4 is indeed induced by the same sort of 9 0 phenolics that induce A. tumefaciens. This level of activity is significantly greater than that observed wi th either W H R or L H R A. tumefaciens strains. Act iv i ty curves for a collection of phenolic compounds are shown i n Figures 4.2-4.7. A range of methoxyphenols were found to be capable of st imulating virulence i n W H R A. tumefaciens (Spencer and Towers, 1988). Here, A 4 / p S M 3 5 8 c d was incubated with a number of these same compounds. These include acetosyringone, acetovanillone, vani l l in , syringaldehyde, the acids vanil l ic , syringic, ferulic, sinapic acid and their methyl esters, coniferyl alcohol, and 2',4',4-trihydroxy-3-methoxychalcone. In this way the relative effectiveness of benzaldehydes, benzoic acids and their methy l esters, acetophenones, cinnamic acids and their methyl esters was assessed. In each case, phenolics of guaiacyl r ing substitution were less effective than the corresponding syringyl substituted compounds. Also , i n each case the methyl esters were more effective than the corresponding free acid. The activity of the monolignol coniferyl alcohol and of the chalcone 2',4',4-trihydroxy-3-methoxychalcone were examined. The activity of the monolignol suggests that a carbonyl group is not an absolute requirement for a i r - i n d u c t i o n . The chalcone was tested to determine whether such a structure could activate vir genes in this species as it d i d in A. tumefaciens (Spencer and Towers, 1988). The curve of activity for the chalcone lies to the left of that of A S , indicating a more strongly active structure than that of the acetophenone. The first general conclusion concerning vir gene expression i n A. rhizogenes is that acetosyringone and other common plant phenolics are capable of inducing the sort of response observed w i t h W H R and L H R A. tumefaciens strains. Second, there are significant differences i n the absolute level of induction recorded i n these three Agrobacterium strains. The units 9 1 Figure 4.2 vir gene activation i n A4/pSM358cd by acetophenones A S and A V 9 2 (0 E 3 0 > u ta © '5 o u ro (0 O) 8000 6000 h 4000 h 2000 r-• AS • Sina pate A Ferulate A Syringate • Van i I late Concentration (uM) Figure 4.3 afr gene activation i n A4/pSM358cd by methoxyphenolic acids 93 8000 • AS • Syringaldehyde A Vanillin Concentration (uM) Figure 4.4 vir gene activation i n A4/pSM358cd by methoxybenzaldehydes 9 4 10000 A AS • Monolignol Concentration (uM) Figure 4.5 vir gene activation i n A4/pSM358cd by coniferyl alcohol 10000 • AS • Chalcone Concentration (uM) Figure 4.6 vir gene activation i n A 4 / p S M 3 5 8 c d by 2' ,4 ' ,4-trihydroxy-3-methoxy chalcone. 9 6 10000 • Me-sinapate • Me-ferulate A Me-syringate A Me-vanillate Concentration (uM) Figure 4.7 vir gene activation in A4/pSM358cd by phenolic methyl esters. 97 of activity recorded i n the strain A4/pSM358cd greatly exceeded that observed i n W H R strain A348. Similarly, at W H R and L H R virB loci , the units of activity recorded i n the strain A348/pSM243cd greatly exceeded that observed i n L H R strain A856/pSM243cd (Spencer et al, 1990). This may be connected w i t h a somewhat longer culture period required by A. rhizogenes strain, or perhaps more stringent conditions of induction. A comparison of the biological activity of. various chemical structures was provided by application of authentic compounds. Phenolics of syr ingyl substi tution are more active than the corresponding guiacyl substituted phenols. This applies to the benzoic acid derivatives vani l l ic and syringic acid, the cinnamic acid derivatives ferulic and sinapic acid (Figure 4.3), and to the aldehydes vani l l in and syringaldehyde (Figure 4.4). The methyl esters of phenolic acids are more active than the free acids (Figure 4.7). For compounds other than the chalcone, the opt imum concentration for air-induction was l O u M or greater. Concentrations as low as 0.1 u M were sufficient for half-maximal induction. The background level of induct ion, sometimes 1000 M i l l e r units , was greater than that observed i n A. tumefaciens strains (Spencer and Towers, 1988). Perhaps this is as a result of preculture of the bacteria in potato-dextrose broth, which may wel l contain active u/r-inducing compounds. This background level of induction d i d not present a problem i n analysis of y/r- induction because these units of 8-galactosidase activity was subtracted from the level of activity f o l l o w i n g induction, to give the induced units of activity. Detectable levels of induct ion were observed w i t h relatively l o w concentrations of inducer compounds compared w i t h those required for vir-induct ion i n A. tumefaciens strains. Thus, for a number of compounds, the 9 8 effective concentration of inducer extends into the nanomolar range. A l o w level of induction was noted by the chalcone at 10 n M . These observations suggest that, while its sensitivity towards efective signal compounds may be diffierent, A. rhizogenes detects the same range of compounds as does A. tumefaciens. It was recently discovered that a naturally occurring chalcone (4,4'-dihydroxy-2'-methoxychalcone) acted along w i t h t w o r e l a t e d f l a v o n o i d s ( 4 ' , 7 - d i h y d r o x y f l a v o n e a n d 4',7-dihydroxyflavanone) i n signall ing Rhizobium meliloti (Maxwell et ah, 1989). Their studies showed that the chalcone was significantly more active than the other f l a v o n o i d s . A s s a y s w i t h an authent ic cha lcone , 2',4',4-trihydroxychalcone, confirmed that chalcones were more active than the f lavanone. The act ivi ty of chalcones i n s ignal l ing A. tumefaciens, and A. rhizogenes as wel l as Rhizobium species, and their relative activity compared w i t h other structures, confirms the close genetic and biochemical relationship of these bacteria. These bacteria appear to have exploited different aspects of the same sort of signalling system. Generally speaking, rhizobia appear to have adapted to the presence of naturally occurring flavonoid root exudates, whereas agrobacteria have adapted to the presence of w o u n d induced methoxy-benzoic and -cinnamic acid derivatives. The experiments described i n this chapter should be fo l lowed by ident i f i ca t ion of natura l ly occurr ing air - inducers of A. rhizogenes i n wounded root exudates. Since a number of azr-activating methoxyphenols have been identified from potato tubers and carrot roots (Chapter 3) it seems very l ike ly that these compounds are naturally occurring A. rhizogenes vir-inducers. 9 9 Chapter 5: GC-mass spectrometry of »tr-inducing mixtures 5.1 Introduction G C - M S (combined gas chromatography-mass spectrometry) is an ideal method for the rapid identification of the phenolic components present i n partially purif ied uzr-inducing mixtures. It offers important advantages over H P L C analysis i n that determinations of chemical structures are not dependent on U V absorption spectra, w h i c h are not diagnostic for specific phenolic compounds even w i t h a photodiode array detector. In comparison, G C retention time and mass spectra, especially of T M S derivatives, provide r igorous proof of a compounds identity. In addi t ion , only very smal l amounts of sample mixtures are required, and a number of components can be identified i n a single G C - M S run. A significant l imitat ion i n G C - M S is the requirement for sample vapor izat ion (Vouros, 1980). Chemical derivi t izat ion is w i d e l y used to increase volatil ity, and can greatly assist mass spectral analysis by generating abundant molecular ions and fragments of diagnostic value. Treatment of s a m p l e s w i t h the d e r i v a t i z i n g a g e n t B S T F A ( b i s -(trimethylsilyl)trifluoroacetamide) i n dry pyridine yields the tr imethylsi lyl (TMS) derivatives of active hydrogens. Concerning s ignal c o m p o u n d mixture analysis, each air-activating structure forms a T M S ether at the phenolic hydroxyl group. In addition, benzoic and cinnamic acids also form T M S esters. The derivat ized mixture of compounds is then separated by gas chromatography (GC) on a fused silica capil lary co lumn. A s the T M S derivatives are eluted, they are fed into the mass spectrometer, ionized by 1 0 0 electron impact (70 eV), and the mass and abundance of the resultant molecular and fragment ions are recorded. The G C retention times and the mass spectra of the compounds are characteristic. 5.2 Methods 5.2.1 Derivatization Aliquots (ca. 400 u.g, i.e. 40 U.L of 10 m g / m L solutions) of active extracts were prepared for BSTFA treatment by first drying small amounts i n micro-"reactivials" on a freeze drying apparatus for at least 24 hrs. W i t h oven dried syringes, 5 u,L of redistilled, dry pyridine, fol lowed by 5 uL of BSTFA (Sigma Co.) was added to each sample. Periodically pyridine was fractionally disti l led, collected over molecular sieves, and stored i n darkness under N 2 to ensure dryness. Heating was not required to achieve derivitization and 1 uL samples were injected directly onto the G C . 5.2.2 G C conditons The G C was fitted with a fused-silica capillary column (30m x 0.25 mm) of SE-54 w i t h H e as the carrier gas. The column was held at 150°C for 2 m i n , programmed at 10°C min" 1 to 300°C and held for 10 min . The injector and zone temp, was 250°C. 1000 scans were accumulated (in 17.30 min.) over each G C run. Compounds of interest were eluted from about 200 to 800 scans ( R t 3.46-13.84 min.). 5.2.3 Mass spectrometer The mass spectrometer component of the combined G C - M S was a F innigan automated M S model 1020 operated i n the electron-ionization 101 mode (70 eV) w i t h an ion source temperature of 95°. A n on line library of mass spectra (National Bureau of Standards) was used, i n conjunction w i t h authentic standards, to assist i n identification of plant-derived compounds. 5.3 Results and Discussion The technique of combined GC-mass spectroscopy was found to be ideal for the identification of phenolic compounds present i n partially purif ied vir - inducing mixtures. O n l y very small quantities of the active fractions were required (although this sometimes required the entire sample), and the components were wel l separated by gas chromatography on the fused-silica capil lary column. W i t h few exceptions, the mass spectrum and retention time of each compound were unique and often these were readily compared w i t h those of an authentic standard. Spectral data accumulated over each G C - M S run were temporarily stored on hard disks (10 M B capacity). Data processing entailed examination of the mass spectrum of each major G C peak, and computer-assisted searches for specified molecular or fragment ions. Because the derivatized phenolics were eluted at predictable times i n each G C run , the number of mass spectra that required examination was greatly reduced. A typical spectrum consisted of a strong M + ion, plus major fragment ions of [M-73] + , [M-15] + , [M-30] + , and often [M-45] + . Mass spectra of interest were compared either w i t h those stored i n an on-line l ibrary (National Bureau of Standards), or w i t h spectra of authentic compounds. Mass spectra of 11 of the uz'r-activating m e t h o x y p h e n o l s observed during the course of this study are shown i n Figures 5.1-5.11. These are presented i n order of acetophenones, a-hydroxyacetophenones, cinnamic 1 0 2 acids, benzoic acids, methyl esters, and the monol ignol coniferyl alcohol. Table 5.1 lists the compounds observed dur ing the course of this survey. These were observed either i n active extracts or were isolated directly from wounded plant tissues. A number of these are very wel l k n o w n ("common") monocycl ic phenolic compounds. Characteristic molecular and fragment ions for a number of key compounds are listed i n Table 5.2. This table also lists the G C R t (by scan number). Table 5.3 lists the molecular weights and molecular ions of various T M S derivatives, and their approximate retention time (by scan number i n a 1000 scan run). The most commonly observed t>i>-inducing compounds i n c l u d e d vanil l ic , syringic, ferulic, sinapic acids (Figures 5.5-5.8). However, a variety of other phenolics were detected i n active mixtures. Traces of vani l l in a n d / o r syringaldehyde were detected i n many samples. The k n o w n virulence inducers, A S and H O - A S were identified as their T M S ethers (Figure 5.1 and 5.2) only i n an extracts from species in the Solanaceae (discussed further i n Chapter 3). A l s o identified were the T M S derivatives of A V and H O - A V (Figure 5.3 and 5.4). The T M S ether of A S produces a very strong M + at m/z 268 , [M-15] + at m/z 253, [M-30J+ at m/z 238, [M-45] + at m/z 223, whereas the T M S ether of A V (30 mass units less than AS) produces a very strong M + at m/z 238 , [M-151+ at m/z 223, [M-30] + at m/z 208, [M-45] + at m/z 193. The two 103 o 4^ 100 -80 -60 -40 20 -200 250 m/z Figure 5.1 Mass spectrum of 3,5-dimethoxy-4-hydroxyacetophenone (TMS derivative) % RA | 100 • 80H 43 60 -I 40 -\ 20 H 59 73 89 0-o I (TMS) OCHg 117 137 1 4 7 165 129 223 193 208 238 —I 250 m / z 50 1 r 100 150 i r 200 Figure 5.2 Mass spectrum of 3-methoxy-4-hydroxyacetophenone (TMS derivative) 253 C H 2 ° ' ( T M S ) ? (TMS) 73 45 5 9 223 8 9 i— i— r 341 356 100 200 - i — i — r - r 300 m/z Figure 5.3 Mass spectrum of a-hydroxy-3>dimethoxy-4-hydroxyacetophenone (TMS derivative) o Figure 5.4 Mass spectrum of a-hydroxy-3-methoxy-4-hydroxyacetophenone (TMS derivative) o 00 Figure 5.5 Mass spectrum of 3,5-dimethoxy-4-hydroxycirinamic acid (TMS derivative) o % RA 100 -8 0 -60 -4 0 -2 0 -73 45 59 338 O Mi (TMS) (TMS) 219 2 4 9 323 191 89 175 145 r*-i—r 100 200 308 293 279 L _ I I 300 m/z Figure 5.6 Mass spectrum of 3-methoxy^-hydroxycinnamic acid (TMS derivative) Figure 5.7 Mass spectrum of 3,5-dimethoxy-4-hydroxybenzoic acid (TMS derivative) % RA 100 -80-60 -40-20-73 45 i — r 59 89 297 0^,0 - (TMS) 1 "'I " I 100 OCH, O I (TMS) 193 223 267 253 137 165 282 312 I I "'I 200 300 m/z Figure 5.8 Mass spectrum of 3-methoxy-4-hydroxybenzoic acid (TMS derivative) 254 O ^ O C H 3 C H 3 0 ' ^ " O C H 3 O (TMS) 269 73 223 284 89 111 L_ 137 195 T 1 1 r 100 T r T r 150 200 I I 250 m/z Figure 5.9 Mass spectrum of methyl-3,5-dimethoxy-4-hydroxybenzoic acid (TMS derivative) 224 73 45 5 9 - r -50 89 107 193 O ^ O C H ) O C H 3 O I (TMS) 137 165 149 179 239 209 I I i i I I I 1 1 r 100 150 T r 200 'I I "" I m/z Figure 5.10 Mass spectrum of methyl-3-methoxy-4-hydroxybenzoic acid (TMS derivative) Figure 5.11 Mass spectrum of coniferyl alcohol (TMS derivative) a - h y d r o x y derivatives, H O - A S and H O - A V (Figure 5.4) each exhibited a strong [M-103] + fragment (base peak) which likely arose as follows: [M-103]+ O ^CH20-(TMS) C H 3 0 ' ^ O C H 3 O (TMS) In fact, H O - A V was identified by mass spectral similarity wi th H O - A S . Both compounds exhibit the [M-103] + ion as their base peak, as wel l as relatively small M + and [M-15l + ions (10-20% R A ) . These mass spectral data are i n agreement w i t h those reported very recently by Klaus Niemela (1990). The kraft pulp ing process generates i n spent (black) l iquor a diverse range of substituted phenols and other compounds, which he sought to identify. W i t h and without derivatization, Niemela identif ied a total of 358 organic compounds i n birch kraft black l iquor , and presented 156 mass spectra, including those of A S , A V , H O - A S , and H O - A V . In addition, he identified the enol tautamers of A S , M + 340, and A V , M+ 310 (Figure 5.12-14). Both Niemela (1990), and Klaus and Spiteller (1989) noted the generation of characteristic [ M - l ] + and [M-31] + ions i n the mass spectra of acetophenone enol tautomers. Perhaps these enol tautomers were present i n the G C - M S samples I examined, but they were overlooked, since the spectra d i d not match any reference spectrum i n the Library used. It w o u l d be interesting to determine the conditions effecting keto-enol 1 1 5 o MeO HO OMe MeO TMS-O B S T F A O 3 / OMe C H , m/z 268 OH M e O y % A C H 2 H O ^ f ^ OMe B S T F A O-TMS M e O y ^ A H z T M S - O ^ f ^ OMe m/z 340 Figure 5.12 Keto-enol tautomerism of acetophenones. T M S derivatives may be used to dist inguish these tautomers: the acetophenone is monotr imethyls i ly la ted , whereas the enol forms a bis(TMS) ether. 1 16 Figure 5.13 Partial mass spectrum of 3/5-dimethoxy-4-hydroxyphenethylenol (TMS) derivative. This tautomer was identified in birch kraft black liquor by GC-MS (Niemela, 1990). AS-enol 300 m/z Figure 5.14 Partial mass spectrum of 4-hydroxy-3-methoxyphenethylenol (TMS) derivative. This AV-enol tautomer was identified in birch kraft black liquor by GC-MS (Niemela, 1990). tautomerism of these acetophenones, and the stability and biological activity of the enol tautomers. N i e m e l a also identif ied 2,6-dimethoxyhydroquinone as its b i s -TMS ether (M+ 314, [M-15] + 299, [M-30] + 284-base peak). Coincidentally, the mass spectrum of an unknown, i n the active V L C fraction f rom Taxus needles happens to closely match the reported mass spectrum of the bis-TMS ether of this quinone (Niemela, 1990). Quinones should.be examined for their effect on growth and virulence induction i n A. tumefaciens. Vir tua l ly every compound had a unique M + ion , w i t h the important exceptions of syringaldehyde, methyl vanillate, and an u n k n o w n isomer, all of M + 254. Fortunately, these can be distinguished on the basis of G C elution profile and mass spectral fragmentation pattern. Authentic syringaldehyde exhibits a strong M + , [M-15] + , [M-30J+, and [M-31]+, but a very small [M-61] + . In contras t , the mass s p e c t r u m of m e t h y l v a n i l l a t e (and that of methylisovanillate) exhibits a strong [M-61] + (m/z 193). Hence the u n k n o w n (Table 5.2, scan #269) may have been methylisovanillate. T w o pairs of phenolic compounds, each exhibiting the same M + ion , were cis and trans . isomers of ferulic and sinapic acids ( M + 338 and 368, respectively). These isomers were wel l separated by G C , w i t h each cis isomer eluting first (Table 5.3). Fatty acids were also observed i n extracts from virtual ly every plant species. A few of these fatty acids are listed, with M + and chemical formulae, i n Table 5.4. The fatty acid mass spectra generally exhibited weak M + ions, and [ M - 1 5 ] + ions roughly 5 times more abundant. Saturated fatty ac id-TMS derivatives had i n common a number of characteristic fragment ions i n c l u d i n g those at m/z 145, 131, 129, and 117. These observations are i n agreement wi th those of Niemela (1990). 119 Based on the k n o w n requirements for vir - induction, it is unlikely that the saturated acids were i n v o l v e d i n s i g n a l l i n g Agrobacterium. Nevertheless, their presence in crude and V L C - p u r i f i e d samples is at least of passing interest. Ferulate esters of higher fatty alcohols are k n o w n from roots of Kalanchoe diagremontiana (Nair et al, 1988). Through growth inhibi t ion bioassays these esters of C22-C30 alcohols were evaluated as allelopathic agents. Interestingly, aside f rom trace quantities of benzaldehydes, no rigorous identification of the active components could be made from G C - M S of a vir-i n d u c i n g extract f rom K. diagremontiana-conditioned m e d i u m . It is conceivable that i n certain cases such esters could be effective s ignal molecules detected by Agrobacterium, but none were detected by G C - M S of active extracts. In summary, the differing compositions of signal compound mixtures detected by G C - M S suggest that the acetophenones are not generally respons ib le for s i g n a l l i n g Agrobacterium (see Chapter 3). Since acetosyringone had not been reported from any plant source prior to its identification as a signal compound from N. tabacum, and since I found a variety of common plant phenolics were capable of i n d u c i n g the same response i n Agrobacterium, I hypothesized that acetosyringone was not l ikely the only naturally occurring signal compound. G C - M S analyses of active samples extracted from media conditioned wi th wounded plant tissues have demonstrated that this is indeed the case. Also , as demonstrated i n Chapter 3, the technique of G C - M S can be employed in signal molecule biosynthetic studies. It is anticipated that future analysis of yfr-activating mixtures w i l l rely, at least i n part, on integrated G C - M S . 120 Table 5.1 Phenolic compounds identified by G C - M S . Chemical name a-hydroxy-3,5-dimethoxy-4-hydroxyacetophenone 3 /5-dimethoxy-4-hydroxyacetophenone 4-hydroxy-3-methoxyacetophenone a-hydroxy-4-hydroxy-3-methoxyacetophenone 4-hydroxyacetophenone 3,5-dimethoxy-4-hydroxybenzaldehyde 3-methoxy-4-hydroxybenzaldehyde 2- hydroxybenzoic acid 3- hydroxybenzoic acid 4- hydroxybenzoic acid 4-hydroxy-3-methoxybenzoic acid 4-hydroxy-3-methoxybenzoic acid, methyl ester 3,5-dimethoxy-4-hydroxybenzoic acid 3,5-dimethoxy-4-hydroxybenzoic acid, methyl ester Z-4-hydroxycinnamic acid E-4-hydroxycinnamic acid 3.4- dihydroxycinnamic acid Z-3,4-dihydroxycinnamic acid, methyl ester E-3,4-dihydroxycinnamic acid, methyl ester Z-4-hydroxy-3-methoxycinnamic acid E-4-hydroxy-3-methoxycinnamic acid E-3,5-dimethoxy-4-hydroxycinnamic acid 3.5- dimethoxy-4-hydroxycinnamyl alcohol C o m m o n name  or abbrevaiation H O - A S Acetosyringone (AS) Acetovanil lone ( A V ) H O - A V Syringaldehyde V a n i l l i n Salicylic acid p -hydroxybenzoic vanil l ic acid vanillic acid methyl ester syringic acid syringic acid methyl ester cis-p -coumaric acid trans-p -coumaric acid caffeic acid cis -methylcaffeiate trans -methylcaffeiate cis -ferulic acid trans -ferulic acid trans -sinapic acid Coniferyl alcohol 121 Table 5.2 Mass spectral data for uzr-inducing phenolics. C o m p o u n d mass % R A v a n i l l i n 224 (29.18) scan # 276 209 (49.64) 194 (100.0) 193 (51.09) syringaldehyde 254 (33.95) scan # 400 239 (45.04) 224 (100.0) 223 (23.70) acetosyringone 268 (42.30) scan # 417 253 (56.64) 238 (70.88) 223 (72.81) H O - A S 356 (14.51) scan # 587 341 (25.37) 253 (100.0) 223 (6.55) 73 (26.87) acetovanil lone 238 (50.52) scan # 323 223 (97.00) 208 (65.70) 193 (100.0) 73 (93.29) H O - A V 326 (6.92) scan # 513 311 (16.96) 223 (84.4) 218 (21.27) 203 (22.07) 175 (18.32) 73 (100.0) syringic acid 342 (69.72) scan # 510 327 (75.60) 312 (68.97) 297 (75.74) 283 (55.28) 122 ferulic acid sinapic acid vanil l ic acid methyl syringate methyl vanillate u n k n o w n 253 (69.94) 223 (60.57) 73 (100.0) 338 (91.22) 323 (82.43) 308 (81.37) 293 (74.81) 279 (45.95) 249 (86.39) 219 (61.10) 73 (100.0) 368 (83.32) 353 (69.57) 338 (91.30) 323 (57.43) 309 (13.25) 279 (54.40) 249 (50.15) 73 (100.0) 312 (54.74) 297 (100.0) 282 (50.94) 267 (61.71) 253 (57.14) 223 (63.47) 193 (54.86) 73 (79.86) 284 (30.38) 269 (43.65) 254 (100.0) 223 (22.36) 73 (31.31) 254 (73.08) 239 (85.41) 224 (100) 193 (86.09) 73 (77.94) 254 (14.74) 239 (1.50) 224 (48.71) scan # 622 scan # 698 scan # 426 scan # 446 scan # 346 scan # 269 123 209 (67.59) 194 (100.0) 193 (64.22) 73 (40.69) 1 2 4 Table 5.3 uz'r-inducing phenolics listed by m.w., M + , and scan # T M S i (~) C o m p o u n d M W m / z scan # A S 196 268 (422) H O - A S 212 356 (587) acetovanil lone 166 248 (323) H O - A V 182 326 (513) Methylsyringate 212 284 (446) V a n i l l i n 152 224 (276) Syringaldehyde 182 254 (400) Vani l l i c acid 168 312 (426) Methylvani l la te 182 254 (346) Syringic acid 198 342 (510) Cinnamic acid 158 230 (327) Caffeic acid 180 252 (576) Ferulic acid 194 338 (619)E (516)Z Sinapic acid 224 368 (698)E (572)Z Methylcaffeate 194 338 (505)E Methylferulate 208 280 (n.d.) Methylsinapate 238 310 (n.d.) Coniferyl alcohol 180 324 (528) Sinapyl alcohol 210 354 (n.d.) p - O H benzoic acid 138 282 (322) o - O H benzoic acid 138 282 (265) p -coumaric acid 164 308 (n.d.) p -OMe-cinnamic 178 250 (438) A v e n a l u m i c acid 190 334 (n.d.) OMe-avenalumic acid 220 364 (n.d.) Zingiberone 194 266 (n.d.) n.d.=not determined 125 Table 5.4 Commonly observed saturated fatty acids i n p/r-inducing mixtures. C o m p o u n d hexadecanoic acid heptadecanoic acid octadecanoic acid dodecanoic acid oleic acid tetradecanoic acid M+ f o r m u l a 328 Ci9H 4o0 2Si 342 ., C2oH4202Si 356 C2lH44C>2Si 272 Ci5H 3 202Si 354 C2iH4202Si 300 Ci9H3602Si 126 Chapter 6: Conclusions 6.1 Survey of Agrobacterium signal compounds Agrobacterium tn'r-inducers are to be found i n , and can be recovered from the organic phase (CH2CI2 or CHCI3 part) of virtually any wounded plant conditioned cell culture medium. Interestingly, the range of plant species w h i c h produce these compounds is not l imited to the normal dicotyledonous hosts. Some hosts exude into the m e d i u m only the common cinnamic or benzoic acid derivatives. Certain others produce benzaldehydes or vir-i n d u c i n g methyl esters. Interestingly, the occurrence of z u r - a c t i v a t i n g acetophenone derivatives appears to be restricted to members of the Solanaceae. In addition, glucosides of active phenolic compounds are present i n unwounded plant tissues. Thus Nicotiana species have small amounts of each of the four acetophenones, as glucosides (see Chapter 3), i n leaf tissue prior to wounding. It was established also that monocots exude into the culture medium a number of the same tn'r-inducing phenolic aglycones as do dicot hosts. The benzoxazinone D I M B O A produced by Zea mays, inhibits growth and virulence of A. tumefaciens and its activity is therefore l ikely responsible for the immuni ty of monocots (such as wheat or maize) to agroinfection. H i g h and l o w D I M B O A strains could be examined for z>ir-inducers and also for D I M B O A content. This may y ie ld important information regarding the susceptibilty at least of certain monocots to transformation by Agrobacterium Other research groups are likely already studying these subjects. Repressors of virulence were noted also i n gymnosperm-conditioned media, and i n the 1 2 7 E t O A c soluble fractions from media conditioned with maize crown tissue and N. glauca stem sections, but these compounds were not identified. Correlations between phenol substitution patterns and induced vir gene activity were previously examined (Spencer and Towers, 1988). Those early results suggested that commonly occurring phenolic compounds may be invo lved i n signall ing Agrobacterium. In the course of the present research, and w i t h an understanding of the secondary plant compounds l i k e l y i n v o l v e d , bioassay-directed pur i f icat ions and G C - M S searches were conducted. G C - M S data confirmed that the suspected phenolics are the active c o m p o u n d s i n extracts. These c o m p o u n d s i n c l u d e the c o m m o n phenylpropanoid acids, benzoic acids, methyl esters, and the acetophenones. Interestingly, one of the first reported signal compounds, A S , was found only i n the active extracts from species within the Solanaceae. In these act ive m i x t u r e s where acetophenones were f o u n d , the c o m m o n phenylpropanoid acids were absent. This could be interpreted as meaning that the C6-C3 acids were the biosynthetic precursors in the pathway to the C6-C2 compounds. A S , H O - A S , A V and H O - A V were produced by a number of species of Nicotiana. This is the first report of the occurrence of A V and H O -A V as signal molecules. H O - A V was tentatively identified by comparison of its mass spectrum w i t h that of H O - A S (described i n Chapter 5), previously synthesized i n our lab (Spencer et al, 1990). The presence of these compounds suggests that the enzyme systems may not be specific to structures w i t h syr ingyl substitution (sinapic acid), but may also use ferulic acid as a substrate. A t least two enzymes would be required to produce the acetophenones from methoxycinnamic acid precursors (Figure 6.1). First, a hydrolyase w o u l d add an equivalent of water to the propenyl double bond. Second, a carbonyl 128 o tl OMe Hydratase O H , M e O ^ / 5 ^ J \ ^ C O O H OMe Oxidase O MeO H O C O O H Decarboxylase OMe CO, O OMe A S Hydroxylase O MeO H O C H 2 O H OMe H O - A S Figure 6.1 Hypothesized pathway to the acetophenones i n Solanaceous plants. 129 function w o u l d be formed at the new (3-hydroxy group to y ie ld the 8-keto acid. Decarboxylation could occur spontaneously, or by the action of a third enzyme, to y ie ld the acetophenone. a - H y d r o x y l a t i o n w o u l d require an a d d i t i o n a l enzyme. Thus three or four enzymes, w h i c h direct the phenylpropanoid pool towards acetophenone derivatives, may be rap id ly synthesized u p o n w o u n d i n g . Cycloheximide inhibi t ion of acetophenone production may occur by inhibition of synthesis of these enzymes. Since phenol ic compounds other than the acetophenones were identif ied from non-solanaceous dicot species, it can be concluded that there is no single chemical signal to Agrobacterium. Indeed, the c o m p o u n d syringic acid methyl ester was isolated a significantly active chemical from a number of grapevine cultivars (Chapter 2; Spencer et al, 1990). The l imited host range (LHR) strain A856 responds to methyl syringate i n a s imilar manner to, but w i t h a different concentration opt imum than, the wide host range (WHR) strain A348. As was discussed i n Chapter 2, the presence of the ester i n active mixtures obtained from grapevine cultivars does not provide a simple explanation for host range determination. Other factors, for example differences i n T - D N A , virC functions, the p H of the condit ioned m e d i u m , occurence of b i n d i n g sites or types of saccharides produced, must play a role in limitation of host range. Perhaps it w i l l be found that the majority of monocot species elaborate the incorrect monosaccharides fol lowing wounding. 6.2 Glucoside precursors and the effects of cycloheximide The biosynthesis of signal compounds was briefly examined. The effects of glucosidase and cycloheximide on production of these compounds 130 were assessed. Glucosides were identified i n intact leaf tissue, but not i n concentrations sufficient to account for the amounts of aglycones recoverable f rom conditioned media. Thus the production of the acetophenones upon w o u n d i n g cannot be explained solely by the action of B-glucosidase activity. Stachel et al. (1985) reported that cycloheximide inhibi ted product ion of acetosyr ingone i n N. tabacum. A s described i n Chapter 3, 20u.M cycloheximide was added to determine its effect on the biosynthesis of inducer compounds from Nicotiana species. Subsequent G C - M S analysis of the active fraction sti l l indicated the presence of smal l amounts of the acetophenone mixture. We interpret this to mean that acetophenone aglycones can be liberated from the glucosides by the action of pre-existing glucosidases, but that synthesis of enzymes is required for product ion of greater levels of signal compounds from phenylpropanoid precursors. Threl fa l l and Whitehead (1988) reported that cellulase treatment i n d u c e d p r o d u c t i o n of acetosyringone. It remains unclear whether acetophenones can be liberated by cellulase from cell walls. It seems more l ike ly that cellulase treatment may elicit de novo synthesis of acetosyringone by mimick ing wounding. 6.3 p H changes i n conditioned media The init ial p H of the M S culture medium was adjusted to 5.70. N o additional buffer was added to this medium. Immediately fol lowing 24 hours incubation at ca. 26 C and 100 rpm, the p H was recorded wi th a p H meter.-Table 3.1 lists the final p H values for a range of species. It is not possible to make generalized conclusions regarding hosts and nonhosts on the basis of p H f rom the data. Nonhost tissues, such as those f rom conifers and 13 1 monocots, can significantly lower the p H of the medium. However , so can certain wounded host tissues such as Nicotiana stem tissue versus leaf tissue. 6.4 vir expression i n Agrobacterium rhizogenes A 4 / p S M 3 5 8 c d Positive results from 13-galactosidase assays w i t h induced cultures of the recipient strain indicated that triparental mating between donor (E. coli pSM358cd), helper (E. coli p R K 2013) and recipient (A. rhizogenes A4) was successful. It was found that under appropriate conditions, an Agrobacterium rhizogenes A 4 background was capable of inducing (3-galactosidase activity from the virEv.lacZ gene fusion reporter plasmid pSM358cd. A brief structure-activity analysis of methoxyphenol-induced virulence i n A. rhizogenes was conducted. A. rhizogenes A 4 responds to u M quantities of acetosyringone and to other • s t r u c t u r a l l y related phenol ics . For tunate ly , the d e r i v a t i v e A 4 / p S M 3 5 8 c d grows and is induced very wel l under the conditions used for analysis of vir gene expression i n A. tumefaciens. However , Agrobacterium rhizogenes A 4 / p S M 3 5 8 c d does not respond to the phenolics i n exactly the same w a y as does A. tumefaciens. A structure-activity analysis of a i r -induction was presented and discussed i n Chapter 4. The background level of A 4 / p S M 3 5 8 c d and its sensitivity to the phenolic compounds is different than i n A. tumefaciens. 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